Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2.

A safe, effective, and scalable vaccine is needed to halt the ongoing SARS-CoV-2 pandemic. We describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 SARS-CoV-2 spike receptor-binding domains (RBDs) in a highly immunogenic array and induce neutralizing antibody titers 10-fold higher than the prefusion-stabilized spike despite a 5-fold lower dose. Antibodies elicited by the RBD nanoparticles target multiple distinct epitopes, suggesting they may not be easily susceptible to escape mutations, and exhibit a lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the assembled nanoparticles suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms and have launched cGMP manufacturing efforts to advance the SARS-CoV-2-RBD nanoparticle vaccine into the clinic.

Systematic analysis of Kaposi's sarcoma (KS)-associated herpesvirus genomes from a KS case-control study in Cameroon: Evidence of dual infections but no association between viral sequence variation and KS risk.

In sub-Saharan Africa, Kaposi's sarcoma-associated herpesvirus (KSHV) is endemic, and Kaposi's sarcoma (KS) is a significant public health problem. Until recently, KSHV genotype analysis was performed using variable gene regions, representing a small fraction of the genome, and thus the contribution of sequence variation to viral transmission or pathogenesis are understudied. We performed near full-length KSHV genome sequence analysis on samples from 43 individuals selected from a large Cameroonian KS case-control study. KSHV genomes were obtained from 21 KS patients and 22 control participants. Phylogenetic analysis of the K1 region indicated the majority of sequences were A5 or B1 subtypes and all three K15 alleles were represented. Unique polymorphisms in the KSHV genome were observed including large gene deletions. We found evidence of multiple distinct KSHV genotypes in three individuals. Additionally, our analyses indicate that recombination is prevalent suggesting that multiple KSHV infections may not be uncommon overall. Most importantly, a detailed analysis of KSHV genomes from KS patients and control participants did not find a correlation between viral sequence variations and disease. Our study is the first to systematically compare near full-length KSHV genome sequences between KS cases and controls in the same endemic region to identify possible sequence variations associated with disease risk.

Sequence Variation of Epstein-Barr Virus: Viral Types, Geography, Codon Usage, and Diseases.

One hundred thirty-eight new Epstein-Barr virus (EBV) genome sequences have been determined. One hundred twenty-five of these and 116 from previous reports were combined to produce a multiple-sequence alignment of 241 EBV genomes, which we have used to analyze variation within the viral genome. The type 1/type 2 classification of EBV remains the major form of variation and is defined mostly by EBNA2 and EBNA3, but the type 2 single-nucleotide polymorphisms (SNPs) at the EBNA3 locus extend into the adjacent gp350 and gp42 genes, whose products mediate infection of B cells by EBV. A small insertion within the BART microRNA region of the genome was present in 21 EBV strains. EBV from saliva of U.S. patients with chronic active EBV infection aligned with the wild-type EBV genome with no evidence of WZhet rearrangements. The V3 polymorphism in the Zp promoter for BZLF1 was found to be frequent in nasopharyngeal carcinoma cases from both Hong Kong and Indonesia. Codon usage was found to differ between latent and lytic cycle EBV genes, and the main forms of variation of the EBNA1 protein have been identified.IMPORTANCE Epstein-Barr virus causes most cases of infectious mononucleosis and posttransplant lymphoproliferative disease. It contributes to several types of cancer, including Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B cell lymphoma, nasopharyngeal carcinoma, and gastric carcinoma. EBV genome variation is important because some of the diseases associated with EBV have very different incidences in different populations and geographic regions, and differences in the EBV genome might contribute to these diseases. Some specific EBV genome alterations that appear to be significant in EBV-associated cancers are already known, and current efforts to make an EBV vaccine and antiviral drugs should also take account of sequence differences in the proteins used as targets.

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.

Heterogeneity of the Epstein-Barr Virus (EBV) Major Internal Repeat Reveals Evolutionary Mechanisms of EBV and a Functional Defect in the Prototype EBV Strain B95-8.

Epstein-Barr virus (EBV) is a ubiquitous pathogen of humans that can cause several types of lymphoma and carcinoma. Like other herpesviruses, EBV has diversified through both coevolution with its host and genetic exchange between virus strains. Sequence analysis of the EBV genome is unusually challenging because of the large number and lengths of repeat regions within the virus. Here we describe the sequence assembly and analysis of the large internal repeat 1 of EBV (IR1; also known as the BamW repeats) for more than 70 strains. The diversity of the latency protein EBV nuclear antigen leader protein (EBNA-LP) resides predominantly within the exons downstream of IR1. The integrity of the putative BWRF1 open reading frame (ORF) is retained in over 80% of strains, and deletions truncating IR1 always spare BWRF1. Conserved regions include the IR1 latency promoter (Wp) and one zone upstream of and two within BWRF1. IR1 is heterogeneous in 70% of strains, and this heterogeneity arises from sequence exchange between strains as well as from spontaneous mutation, with interstrain recombination being more common in tumor-derived viruses. This genetic exchange often incorporates regions of <1 kb, and allelic gene conversion changes the frequency of small regions within the repeat but not close to the flanks. These observations suggest that IR1-and, by extension, EBV-diversifies through both recombination and breakpoint repair, while concerted evolution of IR1 is driven by gene conversion of small regions. Finally, the prototype EBV strain B95-8 contains four nonconsensus variants within a single IR1 repeat unit, including a stop codon in the EBNA-LP gene. Repairing IR1 improves EBNA-LP levels and the quality of transformation by the B95-8 bacterial artificial chromosome (BAC).IMPORTANCE Epstein-Barr virus (EBV) infects the majority of the world population but causes illness in only a small minority of people. Nevertheless, over 1% of cancers worldwide are attributable to EBV. Recent sequencing projects investigating virus diversity to see if different strains have different disease impacts have excluded regions of repeating sequence, as they are more technically challenging. Here we analyze the sequence of the largest repeat in EBV (IR1). We first characterized the variations in protein sequences encoded across IR1. In studying variations within the repeat of each strain, we identified a mutation in the main laboratory strain of EBV that impairs virus function, and we suggest that tumor-associated viruses may be more likely to contain DNA mixed from two strains. The patterns of this mixing suggest that sequences can spread between strains (and also within the repeat) by copying sequence from another strain (or repeat unit) to repair DNA damage.

Natural Variation of Epstein-Barr Virus Genes, Proteins, and Primary MicroRNA.

Viral gene sequences from an enlarged set of about 200 Epstein-Barr virus (EBV) strains, including many primary isolates, have been used to investigate variation in key viral genetic regions, particularly LMP1, Zp, gp350, EBNA1, and the BART microRNA (miRNA) cluster 2. Determination of type 1 and type 2 EBV in saliva samples from people from a wide range of geographic and ethnic backgrounds demonstrates a small percentage of healthy white Caucasian British people carrying predominantly type 2 EBV. Linkage of Zp and gp350 variants to type 2 EBV is likely to be due to their genes being adjacent to the EBNA3 locus, which is one of the major determinants of the type 1/type 2 distinction. A novel classification of EBNA1 DNA binding domains, named QCIGP, results from phylogeny analysis of their protein sequences but is not linked to the type 1/type 2 classification. The BART cluster 2 miRNA region is classified into three major variants through single-nucleotide polymorphisms (SNPs) in the primary miRNA outside the mature miRNA sequences. These SNPs can result in altered levels of expression of some miRNAs from the BART variant frequently present in Chinese and Indonesian nasopharyngeal carcinoma (NPC) samples. The EBV genetic variants identified here provide a basis for future, more directed analysis of association of specific EBV variations with EBV biology and EBV-associated diseases.IMPORTANCE Incidence of diseases associated with EBV varies greatly in different parts of the world. Thus, relationships between EBV genome sequence variation and health, disease, geography, and ethnicity of the host may be important for understanding the role of EBV in diseases and for development of an effective EBV vaccine. This paper provides the most comprehensive analysis so far of variation in specific EBV genes relevant to these diseases and proposed EBV vaccines. By focusing on variation in LMP1, Zp, gp350, EBNA1, and the BART miRNA cluster 2, new relationships with the known type 1/type 2 strains are demonstrated, and a novel classification of EBNA1 and the BART miRNAs is proposed.

Network properties derived from deep sequencing of human B-cell receptor repertoires delineate B-cell populations.

The adaptive immune response selectively expands B- and T-cell clones following antigen recognition by B- and T-cell receptors (BCR and TCR), respectively. Next-generation sequencing is a powerful tool for dissecting the BCR and TCR populations at high resolution, but robust computational analyses are required to interpret such sequencing. Here, we develop a novel computational approach for BCR repertoire analysis using established next-generation sequencing methods coupled with network construction and population analysis. BCR sequences organize into networks based on sequence diversity, with differences in network connectivity clearly distinguishing between diverse repertoires of healthy individuals and clonally expanded repertoires from individuals with chronic lymphocytic leukemia (CLL) and other clonal blood disorders. Network population measures defined by the Gini Index and cluster sizes quantify the BCR clonality status and are robust to sampling and sequencing depths. BCR network analysis therefore allows the direct and quantifiable comparison of BCR repertoires between samples and intra-individual population changes between temporal or spatially separated samples and over the course of therapy.

Genome diversity of Epstein-Barr virus from multiple tumor types and normal infection.

UNLABELLED: Epstein-Barr virus (EBV) infects most of the world's population and is causally associated with several human cancers, but little is known about how EBV genetic variation might influence infection or EBV-associated disease. There are currently no published wild-type EBV genome sequences from a healthy individual and very few genomes from EBV-associated diseases. We have sequenced 71 geographically distinct EBV strains from cell lines, multiple types of primary tumor, and blood samples and the first EBV genome from the saliva of a healthy carrier. We show that the established genome map of EBV accurately represents all strains sequenced, but novel deletions are present in a few isolates. We have increased the number of type 2 EBV genomes sequenced from one to 12 and establish that the type 1/type 2 classification is a major feature of EBV genome variation, defined almost exclusively by variation of EBNA2 and EBNA3 genes, but geographic variation is also present. Single nucleotide polymorphism (SNP) density varies substantially across all known open reading frames and is highest in latency-associated genes. Some T-cell epitope sequences in EBNA3 genes show extensive variation across strains, and we identify codons under positive selection, both important considerations for the development of vaccines and T-cell therapy. We also provide new evidence for recombination between strains, which provides a further mechanism for the generation of diversity. Our results provide the first global view of EBV sequence variation and demonstrate an effective method for sequencing large numbers of genomes to further understand the genetics of EBV infection. IMPORTANCE: Most people in the world are infected by Epstein-Barr virus (EBV), and it causes several human diseases, which occur at very different rates in different parts of the world and are linked to host immune system variation. Natural variation in EBV DNA sequence may be important for normal infection and for causing disease. Here we used rapid, cost-effective sequencing to determine 71 new EBV sequences from different sample types and locations worldwide. We showed geographic variation in EBV genomes and identified the most variable parts of the genome. We identified protein sequences that seem to have been selected by the host immune system and detected variability in known immune epitopes. This gives the first overview of EBV genome variation, important for designing vaccines and immune therapy for EBV, and provides techniques to investigate relationships between viral sequence variation and EBV-associated diseases.

Discovery of a polyomavirus in European badgers (Meles meles) and the evolution of host range in the family Polyomaviridae.

Polyomaviruses infect a diverse range of mammalian and avian hosts, and are associated with a variety of symptoms. However, it is unknown whether the viruses are found in all mammalian families and the evolutionary history of the polyomaviruses is still unclear. Here, we report the discovery of a novel polyomavirus in the European badger (Meles meles), which to our knowledge represents the first polyomavirus to be characterized in the family Mustelidae, and within a European carnivoran. Although the virus was discovered serendipitously in the supernatant of a cell culture inoculated with badger material, we subsequently confirmed its presence in wild badgers. The European badger polyomavirus was tentatively named Meles meles polyomavirus 1 (MmelPyV1). The genome is 5187 bp long and encodes proteins typical of polyomaviruses. Phylogenetic analyses including all known polyomavirus genomes consistently group MmelPyV1 with California sea lion polyomavirus 1 across all regions of the genome. Further evolutionary analyses revealed phylogenetic discordance amongst polyomavirus genome regions, possibly arising from evolutionary rate heterogeneity, and a complex association between polyomavirus phylogeny and host taxonomic groups.

Human immunodeficiency virus Tat associates with a specific set of cellular RNAs.

BACKGROUND: Human Immunodeficiency Virus 1 (HIV-1) exhibits a wide range of interactions with the host cell but whether viral proteins interact with cellular RNA is not clear. A candidate interacting factor is the trans-activator of transcription (Tat) protein. Tat is required for expression of virus genes but activates transcription through an unusual mechanism; binding to an RNA stem-loop, the transactivation response element (TAR), with the host elongation factor P-TEFb. HIV-1 Tat has also been shown to alter the expression of host genes during infection, contributing to viral pathogenesis but, whether Tat also interacts with cellular RNAs is unknown. RESULTS: Using RNA immunoprecipitation coupled with microarray analysis, we have discovered that HIV-1 Tat is associated with a specific set of human mRNAs in T cells. mRNAs bound by Tat share a stem-loop structural element and encode proteins with common biological roles. In contrast, we do not find evidence that Tat associates with microRNAs or the RNA-induced silencing complex (RISC). The interaction of Tat with cellular RNA requires an intact RNA binding domain and Tat RNA binding is linked to an increase in RNA abundance in cell lines and during infection of primary CD4+ T cells by HIV. CONCLUSIONS: We conclude that Tat interacts with a specific set of human mRNAs in T cells, many of which show changes in abundance in response to Tat and HIV infection. This work uncovers a previously unrecognised interaction between HIV and its host that may contribute to viral alteration of the host cellular environment.

Capturing needles in haystacks: a comparison of B-cell receptor sequencing methods.

BACKGROUND: Deep-sequencing methods are rapidly developing in the field of B-cell receptor (BCR) and T-cell receptor (TCR) diversity. These promise to revolutionise our understanding of adaptive immune dynamics, identify novel antibodies, and allow monitoring of minimal residual disease. However, different methods for BCR and TCR enrichment and amplification have been proposed. Here we perform the first systematic comparison between different methods of enrichment, amplification and sequencing for generating BCR and TCR repertoires using large sample numbers. RESULTS: Resampling from the same RNA or cDNA pool results in highly correlated and reproducible repertoires, but resampling low frequency clones leads to stochastic variance. Repertoires generated by different sequencing methods (454 Roche and Illumina MiSeq) and amplification methods (multiplex PCR, 5' Rapid amplification of cDNA ends (5'RACE), and RNA-capture) are highly correlated, and resulting IgHV gene frequencies between the different methods were not significantly different. Read length has an impact on captured repertoire structure, and ultimately full-length BCR sequences are most informative for repertoire analysis as diversity outside of the CDR is very useful for phylogenetic analysis. Additionally, we show RNA-based BCR repertoires are more informative than using DNA. CONCLUSIONS: Repertoires generated by different sequencing and amplification methods are consistent, but we show that read lengths, depths and error profiles should be considered in experimental design, and multiple sampling approaches could be employed to minimise stochastic sampling variation. This detailed investigation of immune repertoire sequencing methods is essential for informing basic and clinical research.

Transmission and evolution of the Middle East respiratory syndrome coronavirus in Saudi Arabia: a descriptive genomic study.

BACKGROUND: Since June, 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) has, worldwide, caused 104 infections in people including 49 deaths, with 82 cases and 41 deaths reported from Saudi Arabia. In addition to confirming diagnosis, we generated the MERS-CoV genomic sequences obtained directly from patient samples to provide important information on MERS-CoV transmission, evolution, and origin. METHODS: Full genome deep sequencing was done on nucleic acid extracted directly from PCR-confirmed clinical samples. Viral genomes were obtained from 21 MERS cases of which 13 had 100%, four 85-95%, and four 30-50% genome coverage. Phylogenetic analysis of the 21 sequences, combined with nine published MERS-CoV genomes, was done. FINDINGS: Three distinct MERS-CoV genotypes were identified in Riyadh. Phylogeographic analyses suggest the MERS-CoV zoonotic reservoir is geographically disperse. Selection analysis of the MERS-CoV genomes reveals the expected accumulation of genetic diversity including changes in the S protein. The genetic diversity in the Al-Hasa cluster suggests that the hospital outbreak might have had more than one virus introduction. INTERPRETATION: We present the largest number of MERS-CoV genomes (21) described so far. MERS-CoV full genome sequences provide greater detail in tracking transmission. Multiple introductions of MERS-CoV are identified and suggest lower R0 values. Transmission within Saudi Arabia is consistent with either movement of an animal reservoir, animal products, or movement of infected people. Further definition of the exposures responsible for the sporadic introductions of MERS-CoV into human populations is urgently needed. FUNDING: Saudi Arabian Ministry of Health, Wellcome Trust, European Community, and National Institute of Health Research University College London Hospitals Biomedical Research Centre.

Full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus.

A novel betacoronavirus associated with lethal respiratory and renal complications was recently identified in patients from several countries in the Middle East. We report the deep genome sequencing of the virus directly from a patient's sputum sample. Our high-throughput sequencing yielded a substantial depth of genome sequence assembly and showed the minority viral variants in the specimen. Detailed phylogenetic analysis of the virus genome (England/Qatar/2012) revealed its close relationship to European bat coronaviruses circulating among the bat species of the Vespertilionidae family. Molecular clock analysis showed that the 2 human infections of this betacoronavirus in June 2012 (EMC/2012) and September 2012 (England/Qatar/2012) share a common virus ancestor most likely considerably before early 2012, suggesting the human diversity is the result of multiple zoonotic events.

Marked endotheliotropism of highly pathogenic avian influenza virus H5N1 following intestinal inoculation in cats.

Highly pathogenic avian influenza virus (HPAIV) H5N1 can infect mammals via the intestine; this is unusual since influenza viruses typically infect mammals via the respiratory tract. The dissemination of HPAIV H5N1 following intestinal entry and associated pathogenesis are largely unknown. To assess the route of spread of HPAIV H5N1 to other organs and to determine its associated pathogenesis, we inoculated infected chicken liver homogenate directly into the intestine of cats by use of enteric-coated capsules. Intestinal inoculation of HPAIV H5N1 resulted in fatal systemic disease. The spread of HPAIV H5N1 from the lumen of the intestine to other organs took place via the blood and lymphatic vascular systems but not via neuronal transmission. Remarkably, the systemic spread of the virus via the vascular system was associated with massive infection of endothelial and lymphendothelial cells, resulting in widespread hemorrhages. This is unique for influenza in mammals and resembles the pathogenesis of HPAIV infection in terrestrial poultry. It contrasts with the pathogenesis of systemic disease from the same virus following entry via the respiratory tract, where lesions are characterized mainly by necrosis and inflammation and are associated with the presence of influenza virus antigen in parenchymal, not endothelial cells. The marked endotheliotropism of the virus following intestinal inoculation indicates that the pathogenesis of systemic influenza virus infection in mammals may differ according to the portal of entry.

Evolutionary dynamics of local pandemic H1N1/2009 influenza virus lineages revealed by whole-genome analysis.

Virus gene sequencing and phylogenetics can be used to study the epidemiological dynamics of rapidly evolving viruses. With complete genome data, it becomes possible to identify and trace individual transmission chains of viruses such as influenza virus during the course of an epidemic. Here we sequenced 153 pandemic influenza H1N1/09 virus genomes from United Kingdom isolates from the first (127 isolates) and second (26 isolates) waves of the 2009 pandemic and used their sequences, dates of isolation, and geographical locations to infer the genetic epidemiology of the epidemic in the United Kingdom. We demonstrate that the epidemic in the United Kingdom was composed of many cocirculating lineages, among which at least 13 were exclusively or predominantly United Kingdom clusters. The estimated divergence times of two of the clusters predate the detection of pandemic H1N1/09 virus in the United Kingdom, suggesting that the pandemic H1N1/09 virus was already circulating in the United Kingdom before the first clinical case. Crucially, three clusters contain isolates from the second wave of infections in the United Kingdom, two of which represent chains of transmission that appear to have persisted within the United Kingdom between the first and second waves. This demonstrates that whole-genome analysis can track in fine detail the behavior of individual influenza virus lineages during the course of a single epidemic or pandemic.

A computational method for immune repertoire mining that identifies novel binders from different clonotypes, demonstrated by identifying anti-pertussis toxoid antibodies.

Due to their shared genetic history, antibodies from the same clonotype often bind to the same epitope. This knowledge is used in immune repertoire mining, where known binders are used to search bulk sequencing repertoires to identify new binders. However, current computational methods cannot identify epitope convergence between antibodies from different clonotypes, limiting the sequence diversity of antigen-specific antibodies that can be identified. We describe how the antibody binding site, the paratope, can be used to cluster antibodies with common antigen reactivity from different clonotypes. Our method, paratyping, uses the predicted paratope to identify these novel cross clonotype matches. We experimentally validated our predictions on a pertussis toxoid dataset. Our results show that even the simplest abstraction of the antibody binding site, using only the length of the loops involved and predicted binding residues, is sufficient to group antigen-specific antibodies and provide additional information to conventional clonotype analysis. Abbreviations: BCR: B-cell receptor; CDR: complementarity-determining region; PTx: pertussis toxoid.

Elicitation of potent neutralizing antibody responses by designed protein nanoparticle vaccines for SARS-CoV-2.

A safe, effective, and scalable vaccine is urgently needed to halt the ongoing SARS-CoV-2 pandemic. Here, we describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 copies of the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) in a highly immunogenic array and induce neutralizing antibody titers roughly ten-fold higher than the prefusion-stabilized S ectodomain trimer despite a more than five-fold lower dose. Antibodies elicited by the nanoparticle immunogens target multiple distinct epitopes on the RBD, suggesting that they may not be easily susceptible to escape mutations, and exhibit a significantly lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the protein components and assembled nanoparticles, especially compared to the SARS-CoV-2 prefusion-stabilized S trimer, suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms for inducing potent neutralizing antibody responses and have launched cGMP manufacturing efforts to advance the lead RBD nanoparticle vaccine into the clinic.

Neural cell adhesion molecule stimulates survival of premyelinating oligodendrocytes via the fibroblast growth factor receptor.

Axonal signals are critical in promoting the survival and maturation of oligodendrocytes during myelination, with contact-dependent signals thought to play a key role. However, the exact nature of these signals remains unclear. Neural cell adhesion molecule (NCAM) is expressed by both axons and oligodendrocytes and is ideally localized to transduce signals from the axon. This study sought to investigate the influence of NCAM on premyelinating oligodendrocytes in vitro. Both a soluble molecule comprising the extracellular domain of NCAM and a peptide derived from the fibroblast growth factor receptor (FGFR) binding motif within the first fibronectin domain stimulated a dose-dependent increase in survival of premyelinating oligodendrocytes in vitro. The survival effect was blocked by a mitogen-activated protein kinase (MAPK) inhibitor and an FGFR inhibitor, suggesting that activation of MAPK signalling pathways following interaction with the FGFR is involved in the survival effect of NCAM. Furthermore, NCAM presented in a cellular monolayer induced an increase in radial process outgrowth of oligodendrocyte progenitor cells. These data suggest that NCAM may play a role in axon-oligodendrocyte signalling during myelination, leading to an increase in oligodendrocyte survival and process outgrowth following axonal contact.

Essential role of inverted repeat in Epstein-Barr virus IR-1 in B cell transformation; geographical variation of the viral genome.

Many regions of the Epstein-Barr virus (EBV) genome, repeated and unique sequences, contribute to the geographical variation observed between strains. Here we use a large alignment of curated EBV genome sequences to identify major sites of variation in the genome of type 1 EBV strains; the CAO deletion in latent membrane protein 1 (LMP1) is the most frequent major indel present in the unique regions of EBV strains from various parts of the world. Principal component analysis was used to identify patterns of sequence variation and nucleotide positions in the sequences that can distinguish EBV from some different geographical regions. Viral genome sequence variation also affects interpretation of genetic content; known genes, origins of replication and gene expression control regions explain most of the viral genome but there are still a few sections of unknown function. One of these EBV genome regions contains a large inverted repeat sequence (invR) within the IR-1 major internal repeat array. We deleted this invR sequence and showed that this abolished the ability of the virus to transform human B cells into lymphoblastoid cell lines. This article is part of the theme issue 'Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses'.

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.