Analysis of the B cell receptor repertoire in six immune-mediated diseases.

B cells are important in the pathogenesis of many, and perhaps all, immune-mediated diseases. Each B cell expresses a single B cell receptor (BCR)1, and the diverse range of BCRs expressed by the total B cell population of an individual is termed the 'BCR repertoire'. Our understanding of the BCR repertoire in the context of immune-mediated diseases is incomplete, and defining this could provide new insights into pathogenesis and therapy. Here, we compared the BCR repertoire in systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Crohn's disease, Behçet's disease, eosinophilic granulomatosis with polyangiitis, and immunoglobulin A (IgA) vasculitis by analysing BCR clonality, use of immunoglobulin heavy-chain variable region (IGHV) genes and-in particular-isotype use. An increase in clonality in systemic lupus erythematosus and Crohn's disease that was dominated by the IgA isotype, together with skewed use of the IGHV genes in these and other diseases, suggested a microbial contribution to pathogenesis. Different immunosuppressive treatments had specific and distinct effects on the repertoire; B cells that persisted after treatment with rituximab were predominately isotype-switched and clonally expanded, whereas the inverse was true for B cells that persisted after treatment with mycophenolate mofetil. Our comparative analysis of the BCR repertoire in immune-mediated disease reveals a complex B cell architecture, providing a platform for understanding pathological mechanisms and designing treatment strategies.

Interferon-Induced Protein 44 and Interferon-Induced Protein 44-Like Restrict Replication of Respiratory Syncytial Virus.

Cellular intrinsic immunity, mediated by the expression of an array of interferon-stimulated antiviral genes, is a vital part of host defense. We have previously used a bioinformatic screen to identify two interferon-stimulated genes (ISG) with poorly characterized function, interferon-induced protein 44 (IFI44) and interferon-induced protein 44-like (IFI44L), as potentially being important in respiratory syncytial virus (RSV) infection. Using overexpression systems, CRISPR-Cas9-mediated knockout, and a knockout mouse model, we investigated the antiviral capability of these genes in the control of RSV replication. Overexpression of IFI44 or IFI44L was sufficient to restrict RSV infection at an early time postinfection. Knocking out these genes in mammalian airway epithelial cells increased levels of infection. Both genes express antiproliferative factors that have no effect on RSV attachment but reduce RSV replication in a minigenome assay. The loss of Ifi44 was associated with a more severe infection phenotype in a mouse model of infection. These studies demonstrate a function for IFI44 and IFI44L in controlling RSV infection.IMPORTANCE RSV infects all children under 2 years of age, but only a subset of children get severe disease. We hypothesize that susceptibility to severe RSV necessitating hospitalization in children without predefined risk factors is, in part, mediated at the antiviral gene level. However, there is a large array of antiviral genes, particularly in the ISG family, the mechanism of which is poorly understood. Having previously identified IFI44 and IFI44L as possible genes of interest in a bioinformatic screen, we dissected the function of these two genes in the control of RSV. Through a range of overexpression and knockout studies, we show that the genes are antiviral and antiproliferative. This study is important because IFI44 and IFI44L are upregulated after a wide range of viral infections, and IFI44L can serve as a diagnostic biomarker of viral infection.

Interferon-Induced Transmembrane Protein 1 Restricts Replication of Viruses That Enter Cells via the Plasma Membrane.

The acute antiviral response is mediated by a family of interferon-stimulated genes (ISGs), providing cell-intrinsic immunity. Mutations in genes encoding these proteins are often associated with increased susceptibility to viral infections. One family of ISGs with antiviral function is the interferon-inducible transmembrane proteins (IFITMs), of which IFITM3 has been studied extensively. In contrast, IFITM1 has not been studied in detail. Since IFITM1 can localize to the plasma membrane, we investigated its function with a range of enveloped viruses thought to infect cells by fusion with the plasma membrane. Overexpression of IFITM1 prevented infection by a number of Paramyxoviridae and Pneumoviridae, including respiratory syncytial virus (RSV), mumps virus, and human metapneumovirus (HMPV). IFITM1 also restricted infection with an enveloped DNA virus that can enter via the plasma membrane, herpes simplex virus 1 (HSV-1). To test the importance of plasma membrane localization for IFITM1 function, we identified blocks of amino acids in the conserved intracellular loop (CIL) domain that altered the subcellular localization of the protein and reduced antiviral activity. By screening reported data sets, 12 rare nonsynonymous single nucleotide polymorphisms (SNPs) were identified in human IFITM1, some of which are in the CIL domain. Using an Ifitm1-/- mouse, we show that RSV infection was more severe, thereby extending the range of viruses restricted in vivo by IFITM proteins and suggesting overall that IFITM1 is broadly antiviral and that this antiviral function is associated with cell surface localization.IMPORTANCE Host susceptibility to viral infection is multifactorial, but early control of viruses not previously encountered is predominantly mediated by the interferon-stimulated gene (ISG) family. There are upwards of 300 of these genes, the majority of which do not have a clearly defined function or mechanism of action. The cellular location of these proteins may have an important effect on their function. One ISG located at the plasma membrane is interferon-inducible transmembrane protein 1 (IFITM1). Here we demonstrate that IFITM1 can inhibit infection with a range of viruses that enter via the plasma membrane. Mutant IFITM1 proteins that were unable to localize to the plasma membrane did not restrict viral infection. We also observed for the first time that IFITM1 plays a role in vivo, and Ifitm1-/- mice were more susceptible to viral lung infection. These data contribute to our understanding of how ISGs prevent viral infections.

Genomic diversity of Epstein-Barr virus genomes isolated from primary nasopharyngeal carcinoma biopsy samples.

UNLABELLED: Undifferentiated nasopharyngeal carcinoma (NPC) has a 100% association with Epstein-Barr virus (EBV). However, only three EBV genomes isolated from NPC patients have been sequenced to date, and the role of EBV genomic variations in the pathogenesis of NPC is unclear. We sought to obtain the sequences of EBV genomes in multiple NPC biopsy specimens in the same geographic location in order to reveal their sequence diversity. Three published EBV (B95-8, C666-1, and HKNPC1) genomes were first resequenced using the sequencing workflow of target enrichment of EBV DNA by hybridization, followed by next-generation sequencing, de novo assembly, and joining of contigs by Sanger sequencing. The sequences of eight NPC biopsy specimen-derived EBV (NPC-EBV) genomes, designated HKNPC2 to HKNPC9, were then determined. They harbored 1,736 variations in total, including 1,601 substitutions, 64 insertions, and 71 deletions, compared to the reference EBV. Furthermore, genes encoding latent, early lytic, and tegument proteins and glycoproteins were found to contain nonsynonymous mutations of potential biological significance. Phylogenetic analysis showed that the HKNPC6 and -7 genomes, which were isolated from tumor biopsy specimens of advanced metastatic NPC cases, were distinct from the other six NPC-EBV genomes, suggesting the presence of at least two parental lineages of EBV among the NPC-EBV genomes. In conclusion, much greater sequence diversity among EBV isolates derived from NPC biopsy specimens is demonstrated on a whole-genome level through a complete sequencing workflow. Large-scale sequencing and comparison of EBV genomes isolated from NPC and normal subjects should be performed to assess whether EBV genomic variations contribute to NPC pathogenesis. IMPORTANCE: This study established a sequencing workflow from EBV DNA capture and sequencing to de novo assembly and contig joining. We reported eight newly sequenced EBV genomes isolated from primary NPC biopsy specimens and revealed the sequence diversity on a whole-genome level among these EBV isolates. At least two lineages of EBV strains are observed, and recombination among these lineages is inferred. Our study has demonstrated the value of, and provided a platform for, genome sequencing of EBV.

Chicken interferon-inducible transmembrane protein 3 restricts influenza viruses and lyssaviruses in vitro.

Interferon-inducible transmembrane protein 3 (IFITM3) is an effector protein of the innate immune system. It confers potent, cell-intrinsic resistance to infection by diverse enveloped viruses both in vitro and in vivo, including influenza viruses, West Nile virus, and dengue virus. IFITM3 prevents cytosolic entry of these viruses by blocking complete virus envelope fusion with cell endosome membranes. Although the IFITM locus, which includes IFITM1, -2, -3, and -5, is present in mammalian species, this locus has not been unambiguously identified or functionally characterized in avian species. Here, we show that the IFITM locus exists in chickens and is syntenic with the IFITM locus in mammals. The chicken IFITM3 protein restricts cell infection by influenza A viruses and lyssaviruses to a similar level as its human orthologue. Furthermore, we show that chicken IFITM3 is functional in chicken cells and that knockdown of constitutive expression in chicken fibroblasts results in enhanced infection by influenza A virus. Chicken IFITM2 and -3 are constitutively expressed in all tissues examined, whereas IFITM1 is only expressed in the bursa of Fabricius, gastrointestinal tract, cecal tonsil, and trachea. Despite being highly divergent at the amino acid level, IFITM3 proteins of birds and mammals can restrict replication of viruses that are able to infect different host species, suggesting IFITM proteins may provide a crucial barrier for zoonotic infections.

Estimating reassortment rates in co-circulating Eurasian swine influenza viruses.

Swine have often been considered as a mixing vessel for different influenza strains. In order to assess their role in more detail, we undertook a retrospective sequencing study to detect and characterize the reassortants present in European swine and to estimate the rate of reassortment between H1N1, H1N2 and H3N2 subtypes with Eurasian (avian-like) internal protein-coding segments. We analysed 69 newly obtained whole genome sequences of subtypes H1N1-H3N2 from swine influenza viruses sampled between 1982 and 2008, using Illumina and 454 platforms. Analyses of these genomes, together with previously published genomes, revealed a large monophyletic clade of Eurasian swine-lineage polymerase segments containing H1N1, H1N2 and H3N2 subtypes. We subsequently examined reassortments between the haemagglutinin and neuraminidase segments and estimated the reassortment rates between lineages using a recently developed evolutionary analysis method. High rates of reassortment between H1N2 and H1N1 Eurasian swine lineages were detected in European strains, with an average of one reassortment every 2-3 years. This rapid reassortment results from co-circulating lineages in swine, and in consequence we should expect further reassortments between currently circulating swine strains and the recent swine-origin H1N1v pandemic strain.

The latent nuclear antigen of Kaposi sarcoma-associated herpesvirus targets the retinoblastoma-E2F pathway and with the oncogene Hras transforms primary rat cells.

Kaposi sarcoma-associated herpesvirus (KSHV) is involved in the etiopathogenesis of Kaposi sar-coma and certain lymphoproliferative disorders. Open reading frame (ORF) 73 encodes the main immunogenic latent nuclear antigen (LNA-1) of KSHV. LNA-1 maintains the KSHV episome and tethers the viral genome to chromatin during mitosis. In addition, LNA-1 interacts with p53 and represses its transcriptional activity. Here we show that LNA-1 also interacts with the retinoblastoma protein. LNA-1 transactivated an artificial promoter carrying the cell cycle transcription factor E2F DNA-binding sequences and also upregulated the cyclin E (CCNEI) promoter, but not the B-myb (MYBL2) promoter. LNA-1 overcame the flat-cell phenotype induced by retinoblastoma protein in Saos2 cells. In cooperation with the cellular oncogene Harvey rat sarcoma viral oncogene homolog (Hras), LNA-1 transformed primary rat embryo fibroblasts and rendered them tumorigenic. These findings indicate that LNA-1 acts as a transcription co-factor and may contribute to KSHV-induced oncogenesis by targeting the retinoblastoma protein-E2F transcriptional regulatory pathway.

Resistance to ddI and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase.

Serial human immunodeficiency virus type-1 (HIV-1) isolates were obtained from five individuals with acquired immunodeficiency syndrome (AIDS) who changed therapy to 2',3'-dideoxyinosine (ddI) after at least 12 months of treatment with 3'-azido-3'-deoxythymidine (zidovudine, AZT). The in vitro sensitivity to ddI decreased during the 12 months following ddI initiation, whereas AZT sensitivity increased. Analysis of the reverse transcriptase coding region revealed a mutation associated with reduced sensitivity to ddI. When this mutation was present in the same genome as a mutation known to confer AZT resistance, the isolates showed increased sensitivity to AZT. Analysis of HIV-1 variants confirmed that the ddI resistance mutation alone conferred ddI and 2',3'-dideoxycytidine resistance, and suppressed the effect of the AZT resistance mutation. The use of combination therapy for HIV-1 disease may prevent drug-resistant isolates from emerging.

Whole genome analysis of local Kenyan and global sequences unravels the epidemiological and molecular evolutionary dynamics of RSV genotype ON1 strains.

The respiratory syncytial virus (RSV) group A variant with the 72-nucleotide duplication in the G gene, genotype ON1, was first detected in Kilifi in 2012 and has almost completely replaced circulating genotype GA2 strains. This replacement suggests some fitness advantage of ON1 over the GA2 viruses in Kilifi, and might be accompanied by important genomic substitutions in ON1 viruses. Close observation of such a new virus genotype introduction over time provides an opportunity to better understand the transmission and evolutionary dynamics of the pathogen. We have generated and analysed 184 RSV-A whole-genome sequences (WGSs) from Kilifi (Kenya) collected between 2011 and 2016, the first ON1 genomes from Africa and the largest collection globally from a single location. Phylogenetic analysis indicates that RSV-A circulation in this coastal Kenya location is characterized by multiple introductions of viral lineages from diverse origins but with varied success in local transmission. We identified signature amino acid substitutions between ON1 and GA2 viruses' surface proteins (G and F), polymerase (L), and matrix M2-1 proteins, some of which were positively selected, and thereby provide an enhanced picture of RSV-A diversity. Furthermore, five of the eleven RSV open reading frames (ORFs) (G, F, L, N, and P) formed distinct phylogenetic clusters for the two genotypes. This might suggest that coding regions outside of the most frequently studied G ORF also play a role in the adaptation of RSV to host populations, with the alternative possibility that some of the substitutions are neutral and provide no selective advantage. Our analysis provides insight into the epidemiological processes that define RSV spread, highlights the genetic substitutions that characterize emerging strains, and demonstrates the utility of large-scale WGS in molecular epidemiological studies.

Erratum: Whole genome analysis of local Kenyan and global sequences unravels the epidemiological and molecular evolutionary dynamics of RSV genotype ON1 strains.

[This corrects the article DOI: 10.1093/ve/vey027.][This corrects the article DOI: 10.1093/ve/vey027.].

Current research priorities in chronic fatigue syndrome/myalgic encephalomyelitis: disease mechanisms, a diagnostic test and specific treatments.

Chronic fatigue syndrome (CFS) is an illness characterised by disabling fatigue of at least 6 months duration, which is accompanied by various rheumatological, infectious and neuropsychiatric symptoms. A collaborative study group has been formed to deal with the current areas for development in CFS research--namely, to develop an understanding of the molecular pathogenesis of CFS, to develop a diagnostic test and to develop specific and curative treatments. Various groups have studied the gene expression in peripheral blood of patients with CFS, and from those studies that have been confirmed using polymerase chain reaction (PCR), clearly, the most predominant functional theme is that of immunity and defence. However, we do not yet know the precise gene signature and metabolic pathways involved. Currently, this is being dealt with using a microarray representing 47,000 human genes and variants, massive parallel signature sequencing and real-time PCR. It will be important to ensure that once a gene signature has been identified, it is specific to CFS and does not occur in other diseases and infections. A diagnostic test is being developed using surface-enhanced, laser-desorption and ionisation-time-of-flight mass spectrometry based on a pilot study in which putative biomarkers were identified. Finally, clinical trials are being planned; novel treatments that we believe are important to trial in patients with CFS are interferon-beta and one of the anti-tumour necrosis factor-alpha drugs.

Whole-genome association study of antibody response to Epstein-Barr virus in an African population: a pilot.

Epstein Barr virus (EBV) infects 95% of the global population and is associated with up to 2% of cancers globally. Immunoglobulin G (IgG) antibody levels to EBV have been shown to be heritable and associated with developing malignancies. We, therefore, performed a pilot genome-wide association analysis of anti-EBV IgG traits in an African population, using a combined approach including array genotyping, whole-genome sequencing and imputation to a panel with African sequence data. In 1562 Ugandans, we identify a variant in human leukocyte antigen (HLA)-DQA1, rs9272371 (p = 2.6 × 10-17) associated with anti-EBV nuclear antigen-1 responses. Trans-ancestry meta-analysis and fine-mapping with European-ancestry individuals suggest the presence of distinct HLA class II variants driving associations in Uganda. In addition, we identify four putative, novel, very rare African-specific loci with preliminary evidence for association with anti-viral capsid antigen IgG responses which will require replication for validation. These findings reinforce the need for the expansion of such studies in African populations with relevant datasets to capture genetic diversity.

VIDA: a virus database system for the organization of animal virus genome open reading frames.

VIDA is a new virus database that organizes open reading frames (ORFs) from partial and complete genomic sequences from animal viruses. Currently VIDA includes all sequences from GenBank for Herpesviridae, Coronaviridae and Arteriviridae. The ORFs are organized into homologous protein families, which are identified on the basis of sequence similarity relationships. Conserved sequence regions of potential functional importance are identified and can be retrieved as sequence alignments. We use a controlled taxonomical and functional classification for all the proteins and protein families in the database. When available, protein structures that are related to the families have also been included. The database is available for online search and sequence information retrieval at http://www.biochem.ucl.ac.uk/bsm/virus_database/ VIDA.html.

Eye on the B-ALL: B-cell receptor repertoires reveal persistence of numerous B-lymphoblastic leukemia subclones from diagnosis to relapse.

The strongest predictor of relapse in B-cell acute lymphoblastic leukemia (B-ALL) is the level of persistence of tumor cells after initial therapy. The high mutation rate of the B-cell receptor (BCR) locus allows high-resolution tracking of the architecture, evolution and clonal dynamics of B-ALL. Using longitudinal BCR repertoire sequencing, we find that the BCR undergoes an unexpectedly high level of clonal diversification in B-ALL cells through both somatic hypermutation and secondary rearrangements, which can be used for tracking the subclonal composition of the disease and detect minimal residual disease with unprecedented sensitivity. We go on to investigate clonal dynamics of B-ALL using BCR phylogenetic analyses of paired diagnosis-relapse samples and find that large numbers of small leukemic subclones present at diagnosis re-emerge at relapse alongside a dominant clone. Our findings suggest that in all informative relapsed patients, the survival of large numbers of clonogenic cells beyond initial chemotherapy is a surrogate for inherent partial chemoresistance or inadequate therapy, providing an increased opportunity for subsequent emergence of fully resistant clones. These results frame early cytoreduction as an important determinant of long-term outcome.

Molecular identification of novel viruses.

Viruses are responsible for many of the diseases caused by microbial infection. During the past two decades, approximately 20 new human viruses have been discovered. Many of these new viruses were initially identified using molecular biology techniques, a major advantage of which is the ability to search rapidly for new viruses, known viruses or related, but previously unidentified, members of established virus families in disease samples.

Gene expression in peripheral blood mononuclear cells from patients with chronic fatigue syndrome.

BACKGROUND: Chronic fatigue syndrome (CFS) is a multisystem disease, the pathogenesis of which remains undetermined. AIMS: To test the hypothesis that there are reproducible abnormalities of gene expression in patients with CFS compared with normal healthy persons. METHODS: To gain further insight into the pathogenesis of this disease, gene expression was analysed in peripheral blood mononuclear cells from 25 patients with CFS diagnosed according to the Centers for Disease Control criteria and 25 normal blood donors matched for age, sex, and geographical location, using a single colour microarray representing 9522 human genes. After normalisation, average difference values for each gene were compared between test and control groups using a cutoff fold difference of expression > or = 1.5 and a p value of 0.001. Genes showing differential expression were further analysed using Taqman real time polymerase chain reaction (PCR) in fresh samples. RESULTS: Analysis of microarray data revealed differential expression of 35 genes. Real time PCR confirmed differential expression in the same direction as array results for 16 of these genes, 15 of which were upregulated (ABCD4, PRKCL1, MRPL23, CD2BP2, GSN, NTE, POLR2G, PEX16, EIF2B4, EIF4G1, ANAPC11, PDCD2, KHSRP, BRMS1, and GABARAPL1) and one of which was downregulated (IL-10RA). This profile suggests T cell activation and perturbation of neuronal and mitochondrial function. Upregulation of neuropathy target esterase and eukaryotic translation initiation factor 4G1 may suggest links with organophosphate exposure and virus infection, respectively. CONCLUSION: These results suggest that patients with CFS have reproducible alterations in gene regulation.

Zidovudine resistance predicted by direct detection of mutations in DNA from HIV-infected lymphocytes.

Zidovudine-resistant strains of HIV have recently been isolated from individuals during prolonged treatment. Analysis of the HIV reverse transcriptase (RT) gene from clinical isolates revealed that resistance was due to multiple nucleotide changes conferring specific amino acid substitutions in this enzyme. In order to correlate the degree of resistance with these amino acid changes, we constructed a series of infectious HIV variants with specific combinations of mutations in the RT gene and assessed their sensitivity to zidovudine. The reproducible nature of the mutations seen in clinical isolates has enabled the polymerase chain reaction to be used to identify lesions associated with resistance. This procedure was validated by analysis of sensitive and resistant clinical isolates with RT genes of known DNA sequence. Using a 'double' amplification procedure, zidovudine sensitivity was assessed by direct detection of specific mutations in DNA from peripheral-blood lymphocyte samples. This should make it possible to test large numbers of individuals receiving zidovudine therapy, with the aim of establishing the clinical significance of the resistant isolates.

HIV-1 biological phenotype and the development of zidovudine resistance in relation to disease progression in asymptomatic individuals during treatment.

OBJECTIVE: To determine which parameters are associated with clinical progression during zidovudine treatment of asymptomatic HIV-1-infected individuals. METHODS: Twenty-four initially asymptomatic HIV-1-infected individuals were treated with zidovudine and followed until the development of AIDS or for approximately 3 years. HIV-1 phenotype was determined by cocultivation of patient cells with donor lymphocytes, and by a new assay of direct cocultivation with MT-2 cells. Specific mutations in the HIV-1 reverse transcriptase (RT) gene conferring resistance to zidovudine were detected using a selective polymerase chain reaction. RESULTS: Progression to AIDS was more rapid in individuals harbouring syncytium-inducing (SI) viral isolates or showing a conversion from non-syncytium-inducing (NSI) to SI viral isolates. One out of 20 patients who spent a total of 559 months harbouring an NSI phenotype progressed to AIDS, whereas eight out of 12 patients who spent a total of 223 months harbouring an SI phenotype progressed to AIDS (P < 0.001). There was no significant difference between SI and non-SI isolates in the frequency of five mutations causing zidovudine resistance. However, all SI isolates obtained after 2 years of treatment contained mutations in codons 41 and 215 of the RT gene, whereas only five out of 11 (45%) NSI isolates obtained at that time had this combination of mutations. CONCLUSIONS: Conversion to the SI phenotype cannot be prevented by zidovudine treatment. The presence or appearance of an SI virus heralded disease progression in zidovudine-treated individuals. Further research is required to investigate the relationship between virus phenotype and development of zidovudine resistance.

Human herpesvirus type 8 and Kaposi's sarcoma.

Kaposi's sarcoma-associated herpesvirus or human herpesvirus type 8 (HHV-8) is present in all forms of Kaposi's sarcoma (KS) as well as in primary effusion lymphomas and some cases of Castleman's disease. In KS tissues, HHV-8 is present in endothelial and spindle cells. Current serologic tests suggest that HHV-8 is predominantly found in those at risk of KS and is not as widespread as most other human herpesviruses. HHV-8 encodes various proteins that may play a role in promotion of cellular growth, including cyclin- and G-coupled protein receptor homologues, and anti-apoptotic proteins, including Bcl-2, IL-6 (i.e., interleukin 6), and FLIP (i.e., FLICE inhibitory protein) homologues. In addition, HHV-8 encodes two macrophage inflammatory-like proteins with anti-human immunodeficiency virus and angiogenic potential.

The isolation and characterisation of plant sequences homologous to human hypervariable minisatellites.

We have isolated DNA probes, homologous to the human hypervariable minisatellite sequence 33.15, from the genome of rice (Oryza sativa). These probes are capable of producing a multilocus rice DNA fingerprint. The rice sequence has a tandem repeating structure based on a 12 bp GC-rich repeat which shows homology to its human counterpart. This probe detects up to 30 loci which are at a number of unlinked chromosomal sites. The GC-rich sequence is invariably associated with an open reading frame (ORF) of unknown function. The ORF is probably a member of a small multigene family.