Publications
343 results found
Goodwin P, Kellam P, 2018, Progressing policy at the society
Correia S, Bridges R, Wegner F, et al., 2018, Sequence variation of Epstein-Barr virus: viral types, geography, codon usage and diseases, Journal of Virology, Vol: 92, ISSN: 1098-5514
138 new Epstein-Barr virus (EBV) genome sequences have been determined. 125 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/type2 classification of EBV remains the major form of variation and is defined mostly by EBNA2 and EBNA3, but the type 2 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 miRNA region of the genome was present in 21 EBV strains. EBV from saliva of USA 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 both from 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 post-transplant 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 - 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.
Otieno JR, Kamau EM, Oketch JW, et al., 2018, Whole genome analysis of local Kenyan and global sequences unravels the epidemiological and molecular evolutionary dynamics of RSV genotype ON1 strains, Virus Evolution, Vol: 4, Pages: 1-13, ISSN: 2057-1577
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.
Petrova V, Muir L, McKay P, et al., 2018, Combined influence of B-cell receptor rearrangement and somatic hypermutation on B-cell class-switch fate in health and in chronic lymphocytic leukaemia, Frontiers in Immunology, Vol: 9, ISSN: 1664-3224
A diverse B-cell receptor (BCR) repertoire is required to bind a wide range of antigens.BCRs are generated through genetic recombination and can be diversified throughsomatic hypermutation (SHM) or class-switch recombination (CSR). Patterns of repertoirediversity can vary substantially between different health conditions. We use isotyperesolvedBCR sequencing to compare B-cell evolution and class-switch fate in healthyindividuals and in patients with chronic lymphocytic leukemia (CLL). We show that thepatterns of SHM and CSR in B-cells from healthy individuals are distinct from CLL. Weidentify distinct properties of clonal expansion that lead to the generation of antibodiesof different classes in healthy, malignant, and non-malignant CLL BCR repertoires. Wefurther demonstrate that BCR diversity is affected by relationships between antibodyvariable and constant regions leading to isotype-specific signatures of variable geneusage. This study provides powerful insights into the mechanisms underlying the evolutionof the adaptive immune responses in health and their aberration during disease.
Goldhill DH, Langat P, Xie H, et al., 2018, Determining the mutation bias of favipiravir in influenza using next-generation sequencing, Publisher: Cold Spring Harbor Laboratory
Favipiravir is a broad spectrum antiviral drug that may be used to treat influenza. Previous research has identified that favipiravir likely acts as a mutagen but the precise mutation bias that favipiravir induces in influenza virus RNAs has not been described. Here, we use next-generation sequencing (NGS) with barcoding of individual RNA molecules to accurately and quantitatively detect favipiravir-induced mutations and to sample orders of magnitude more mutations than would be possible through Sanger sequencing. We demonstrate that favipiravir causes mutations and show that favipiravir primarily acts as a guanine analogue and secondarily as an adenine analogue resulting in the accumulation of transition mutations. We also use a standard NGS pipeline to show that the mutagenic effect of favipiravir can be measured by whole genome sequencing of virus.
Phan MVT, Tue NT, Pham HA, et al., 2018, Identification and characterization of Coronaviridae genomes from Vietnamese bats and rats based on conserved protein domains, Virus Evolution, Vol: 4, ISSN: 2057-1577
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.
Otieno JR, Kamau EM, Oketch JW, et al., 2018, Corrigendum: Whole genome analysis of local Kenyan and global sequences unravels the epidemiological and molecular evolutionary dynamics of RSV genotype ON1 strains, Virus Evolution, Vol: 4, ISSN: 2057-1577
This is a correction to: Virus Evolution, Volume 4, Issue 2, July 2018, vey027, https://doi.org/10.1093/ve/vey027
Kiyuka PK, Agoti CN, Munywoki PK, et al., 2018, Human coronavirus NL63 molecular epidemiology and evolutionary patterns in rural coastal Kenya, Journal of Infectious Diseases, Vol: 217, Pages: 1728-1739, ISSN: 0022-1899
BackgroundHuman coronavirus NL63 (HCoV-NL63) is a globally endemic pathogen causing mild and severe respiratory tract infections with reinfections occurring repeatedly throughout a lifetime.MethodsNasal samples were collected in coastal Kenya through community-based and hospital-based surveillance. HCoV-NL63 was detected with multiplex real-time reverse transcription PCR, and positive samples were targeted for nucleotide sequencing of the spike (S) protein. Additionally, paired samples from 25 individuals with evidence of repeat HCoV-NL63 infection were selected for whole-genome virus sequencing.ResultsHCoV-NL63 was detected in 1.3% (75/5573) of child pneumonia admissions. Two HCoV-NL63 genotypes circulated in Kilifi between 2008 and 2014. Full genome sequences formed a monophyletic clade closely related to contemporary HCoV-NL63 from other global locations. An unexpected pattern of repeat infections was observed with some individuals showing higher viral titers during their second infection. Similar patterns for 2 other endemic coronaviruses, HCoV-229E and HCoV-OC43, were observed. Repeat infections by HCoV-NL63 were not accompanied by detectable genotype switching.ConclusionsIn this coastal Kenya setting, HCoV-NL63 exhibited low prevalence in hospital pediatric pneumonia admissions. Clade persistence with low genetic diversity suggest limited immune selection, and absence of detectable clade switching in reinfections indicates initial exposure was insufficient to elicit a protective immune response.
Dunning J, Blankley S, Hoang LT, et al., 2018, Progression of whole-blood transcriptional signatures from interferon-induced to neutrophil-associated patterns in severe influenza, Nature Immunology, Vol: 19, Pages: 625-635, ISSN: 1529-2916
Transcriptional profiles and host-response biomarkers are used increasingly to investigate the severity, subtype and pathogenesis of disease. We now describe whole-blood mRNA signatures and concentrations of local and systemic immunological mediators in 131 adults hospitalized with influenza, from whom extensive clinical and investigational data were obtained by MOSAIC investigators. Signatures reflective of interferon-related antiviral pathways were common up to day 4 of symptoms in patients who did not require mechanical ventilator support; in those who needed mechanical ventilation, an inflammatory, activated-neutrophil and cell-stress or death (‘bacterial’) pattern was seen, even early in disease. Identifiable bacterial co-infection was not necessary for this ‘bacterial’ signature but was able to enhance its development while attenuating the early ‘viral’ signature. Our findings emphasize the importance of timing and severity in the interpretation of host responses to acute viral infection and identify specific patterns of immune-system activation that might enable the development of novel diagnostic and therapeutic tools for severe influenza.
Otieno J, Kamau E, Oketch J, et al., 2018, Whole genome analysis of local Kenyan and global sequences unravels the epidemiological and molecular evolutionary dynamics of RSV genotype ON1 strains, Publisher: bioRxiv
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 previously circulating genotype GA2 strains. This replacement suggests some fitness advantage of ON1 over the GA2 viruses, and might be accompanied by important genomic substitutions in ON1 viruses. Close observation of such a new virus introduction over time provides an opportunity to better understand the transmission and evolutionary dynamics of the pathogen. We have generated and analyzed 184 RSV-A whole genome sequences (WGS) 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 transmission into this coastal Kenya location is characterized by multiple introductions of viral lineages from diverse origins but with varied success in local transmission. We identify signature amino acid substitutions between ON1 and GA2 viruses within genes encoding the surface proteins (G, F), polymerase (L) and matrix M2-1 proteins, some of which were identified as positively selected, and thereby provide an enhanced picture of RSV-A diversity. Furthermore, five of the eleven RSV open reading frames (ORF) (i.e. G, F, L, N and P), analyzed separately, formed distinct phylogenetic clusters for the two genotypes. This might suggest that coding regions outside of the most frequently studied G ORF play a role in the adaptation of RSV to host populations with the alternative possibility that some of the substitutions are nothing more than genetic hitchhikers. 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.
Van Nguyen D, Van Nguyen C, Bonsall D, et al., 2018, Detection and characterization of homologues of human hepatitis vruses and pegiviruses in rodents and bats in Vietnam, Viruses, Vol: 10, ISSN: 1999-4915
Rodents and bats are now widely recognised as important sources of zoonotic virus infections in other mammals, including humans. Numerous surveys have expanded our knowledge of diverse viruses in a range of rodent and bat species, including their origins, evolution, and range of hosts. In this study of pegivirus and human hepatitis-related viruses, liver and serum samples from Vietnamese rodents and bats were examined by PCR and sequencing. Nucleic acids homologous to human hepatitis B, C, E viruses were detected in liver samples of 2 (1.3%) of 157 bats, 38 (8.1%), and 14 (3%) of 470 rodents, respectively. Hepacivirus-like viruses were frequently detected (42.7%) in the bamboo rat, Rhizomys pruinosus, while pegivirus RNA was only evident in 2 (0.3%) of 638 rodent serum samples. Complete or near-complete genome sequences of HBV, HEV and pegivirus homologues closely resembled those previously reported from rodents and bats. However, complete coding region sequences of the rodent hepacivirus-like viruses substantially diverged from all of the currently classified variants and potentially represent a new species in the Hepacivirus genus. Of the viruses identified, their routes of transmission and potential to establish zoonoses remain to be determined.
Berto A, Anh PH, Carrique-Mas JJ, et al., 2018, Detection of potentially novel paramyxovirus and coronavirus viral RNA in bats and rats in the Mekong Delta region of southern Viet Nam, Zoonoses and Public Health, Vol: 65, Pages: 30-42, ISSN: 0931-1793
Bats and rodents are being increasingly recognized as reservoirs of emerging zoonotic viruses. Various studies have investigated bat viruses in tropical regions, but to date there are no data regarding viruses with zoonotic potential that circulate in bat and rat populations in Viet Nam. To address this paucity of data, we sampled three bat farms and three wet markets trading in rat meat in the Mekong Delta region of southern Viet Nam. Faecal and urine samples were screened for the presence of RNA from paramyxoviruses, coronaviruses and filoviruses. Paramyxovirus RNA was detected in 4 of 248 (1%) and 11 of 222 (4.9%) bat faecal and urine samples, respectively. Coronavirus RNA was detected in 55 of 248 (22%) of bat faecal samples; filovirus RNA was not detected in any of the bat samples. Further, coronavirus RNA was detected in 12 of 270 (4.4%) of rat faecal samples; all samples tested negative for paramyxovirus. Phylogenetic analysis revealed that the bat paramyxoviruses and bat and rat coronaviruses were related to viruses circulating in bat and rodent populations globally, but showed no cross‐species mixing of viruses between bat and rat populations within Viet Nam. Our study shows that potentially novel variants of paramyxoviruses and coronaviruses commonly circulate in bat and rat populations in Viet Nam. Further characterization of the viruses and additional human and animal surveillance is required to evaluate the likelihood of viral spillover and to assess whether these viruses pose a risk to human health.
Berto A, Pham HA, Thao TTN, et al., 2018, Hepatitis E in southern Vietnam: Seroepidemiology in humans and molecular epidemiology in pigs, Zoonoses and Public Health, Vol: 65, Pages: 43-50, ISSN: 0931-1793
Viral pathogens account for a significant proportion of the burden of emerging infectious diseases in humans. The Wellcome Trust‐Vietnamese Initiative on Zoonotic Infections (WT‐VIZIONS) is aiming to understand the circulation of viral zoonotic pathogens in animals that pose a potential risk to human health. Evidence suggests that human exposure and infections with hepatitis E virus (HEV) genotypes (GT) 3 and 4 results from zoonotic transmission. Hypothesising that HEV GT3 and GT4 are circulating in the Vietnamese pig population and can be transmitted to humans, we aimed to estimate the seroprevalence of HEV exposure in a population of farmers and the general population. We additionally performed sequence analysis of HEV in pig populations in the same region to address knowledge gaps regarding HEV circulation and to evaluate if pigs were a potential source of HEV exposure. We found a high prevalence of HEV GT3 viral RNA in pigs (19.1% in faecal samples and 8.2% in rectal swabs) and a high HEV seroprevalence in pig farmers (16.0%) and a hospital‐attending population (31.7%) in southern Vietnam. The hospital population was recruited as a general‐population proxy even though this particular population subgroup may introduce bias. The detection of HEV RNA in pigs indicates that HEV may be a zoonotic disease risk in this location, although a larger sample size is required to infer an association between HEV positivity in pigs and seroprevalence in humans.
Yebra G, Frampton D, Cassarino TG, et al., 2018, A high HIV-1 strain variability in London, UK, revealed by full-genome analysis: Results from the ICONIC project, PLoS ONE, Vol: 13, ISSN: 1932-6203
Background & methodsThe ICONIC project has developed an automated high-throughput pipeline to generate HIV nearly full-length genomes (NFLG, i.e. from gag to nef) from next-generation sequencing (NGS) data. The pipeline was applied to 420 HIV samples collected at University College London Hospitals NHS Trust and Barts Health NHS Trust (London) and sequenced using an Illumina MiSeq at the Wellcome Trust Sanger Institute (Cambridge). Consensus genomes were generated and subtyped using COMET, and unique recombinants were studied with jpHMM and SimPlot. Maximum-likelihood phylogenetic trees were constructed using RAxML to identify transmission networks using the Cluster Picker.ResultsThe pipeline generated sequences of at least 1Kb of length (median = 7.46Kb, IQR = 4.01Kb) for 375 out of the 420 samples (89%), with 174 (46.4%) being NFLG. A total of 365 sequences (169 of them NFLG) corresponded to unique subjects and were included in the down-stream analyses. The most frequent HIV subtypes were B (n = 149, 40.8%) and C (n = 77, 21.1%) and the circulating recombinant form CRF02_AG (n = 32, 8.8%). We found 14 different CRFs (n = 66, 18.1%) and multiple URFs (n = 32, 8.8%) that involved recombination between 12 different subtypes/CRFs. The most frequent URFs were B/CRF01_AE (4 cases) and A1/D, B/C, and B/CRF02_AG (3 cases each). Most URFs (19/26, 73%) lacked breakpoints in the PR+RT pol region, rendering them undetectable if only that was sequenced. Twelve (37.5%) of the URFs could have emerged within the UK, whereas the rest were probably imported from sub-Saharan Africa, South East Asia and South America. For 2 URFs we found highly similar pol sequences circulating in the UK. We detected 31 phylogenetic clusters using the full dataset: 25 pairs (mostly subtypes B and C), 4 triplets and 2 quadruplets. Some of these were not consistent across different genes due to inter- and intra-subtype recombination. Clusters involved 70 sequences, 19.2% of the dataset.ConclusionsT
Groves HT, McDonald JU, Langat P, et al., 2018, Mouse Models of Influenza Infection with Circulating Strains to Test Seasonal Vaccine Efficacy, Frontiers in Immunology, Vol: 9, ISSN: 1664-3224
Influenza virus infection is a significant cause of morbidity and mortality worldwide. The surface antigens of influenza virus change over time blunting both naturally acquired and vaccine induced adaptive immune protection. Viral antigenic drift is a major contributing factor to both the spread and disease burden of influenza. The aim of this study was to develop better infection models using clinically relevant, influenza strains to test vaccine induced protection. CB6F1 mice were infected with a range of influenza viruses and disease, inflammation, cell influx, and viral load were characterized after infection. Infection with circulating H1N1 and representative influenza B viruses induced a dose-dependent disease response; however, a recent seasonal H3N2 virus did not cause any disease in mice, even at high titers. Viral infection led to recoverable virus, detectable both by plaque assay and RNA quantification after infection, and increased upper airway inflammation on day 7 after infection comprised largely of CD8 T cells. Having established seasonal infection models, mice were immunized with seasonal inactivated vaccine and responses were compared to matched and mismatched challenge strains. While the H1N1 subtype strain recommended for vaccine use has remained constant in the seven seasons between 2010 and 2016, the circulating strain of H1N1 influenza (2009 pandemic subtype) has drifted both genetically and antigenically since 2009. To investigate the effect of this observed drift on vaccine induced protection, mice were immunized with antigens from A/California/7/2009 (H1N1) and challenged with H1N1 subtype viruses recovered from 2009, 2010, or 2015. Vaccination with A/California/7/2009 antigens protected against infection with either the 2009 or 2010 strains, but was less effective against the 2015 strain. This observed reduction in protection suggests that mouse models of influenza virus vaccination and infection can be used as an additional tool to predic
Muir L, McKay PF, Petrova VN, et al., 2018, Optimisation ofex vivomemory B cell expansion/differentiation for interrogation of rare peripheral memory B cell subset responses [version 2; referees: 2 approved], Wellcome Open Research, Vol: 2, Pages: 97-97, ISSN: 2398-502X
Background: Human memory B cells play a vital role in the long-term protection of the host from pathogenic re-challenge. In recent years the importance of a number of different memory B cell subsets that can be formed in response to vaccination or infection has started to become clear. To study memory B cell responses, cells can be culturedex vivo,allowing for an increase in cell number and activation of these quiescent cells, providing sufficient quantities of each memory subset to enable full investigation of functionality. However, despite numerous papers being published demonstrating bulk memory B cell culture, we could find no literature on optimised conditions for the study of memory B cell subsets, such as IgM+memory B cells. Methods:Following a literature review, we carried out a large screen of memory B cell expansion conditions to identify the combination that induced the highest levels of memory B cell expansion. We subsequently used a novel Design of Experiments approach to finely tune the optimal memory B cell expansion and differentiation conditions for human memory B cell subsets. Finally, we characterised the resultant memory B cell subpopulations by IgH sequencing and flow cytometry. Results:The application of specific optimised conditions induce multiple rounds of memory B cell proliferation equally across Ig isotypes, differentiation of memory B cells to antibody secreting cells, and importantly do not alter the Ig genotype of the stimulated cells. Conclusions:Overall, our data identify a memory B cell culture system that offers a robust platform for investigating the functionality of rare memory B cell subsets to infection and/or vaccination.
Brener J, Gall A, Hurst J, et al., 2018, Rapid HIV disease progression following superinfection in an HLA-B*27:05/B*57:01-positive transmission recipient, Retrovirology, Vol: 15, ISSN: 1742-4690
BackgroundThe factors determining differential HIV disease outcome among individuals expressing protective HLA alleles such as HLA-B*27:05 and HLA-B*57:01 remain unknown. We here analyse two HIV-infected subjects expressing both HLA-B*27:05 and HLA-B*57:01. One subject maintained low-to-undetectable viral loads for more than a decade of follow up. The other progressed to AIDS in < 3 years.ResultsThe rapid progressor was the recipient within a known transmission pair, enabling virus sequences to be tracked from transmission. Progression was associated with a 12% Gag sequence change and 26% Nef sequence change at the amino acid level within 2 years. Although next generation sequencing from early timepoints indicated that multiple CD8+ cytotoxic T lymphocyte (CTL) escape mutants were being selected prior to superinfection, < 4% of the amino acid changes arising from superinfection could be ascribed to CTL escape. Analysis of an HLA-B*27:05/B*57:01 non-progressor, in contrast, demonstrated minimal virus sequence diversification (1.1% Gag amino acid sequence change over 10 years), and dominant HIV-specific CTL responses previously shown to be effective in control of viraemia were maintained. Clonal sequencing demonstrated that escape variants were generated within the non-progressor, but in many cases were not selected. In the rapid progressor, progression occurred despite substantial reductions in viral replicative capacity (VRC), and non-progression in the elite controller despite relatively high VRC.ConclusionsThese data are consistent with previous studies demonstrating rapid progression in association with superinfection and that rapid disease progression can occur despite the relatively the low VRC that is typically observed in the setting of multiple CTL escape mutants.
Wymant C, Blanquart F, Golubchik T, et al., 2018, Easy and accurate reconstruction of whole HIV genomes from short-read sequence data with shiver, Virus Evolution, Vol: 4, ISSN: 2057-1577
Studying the evolution of viruses and their molecular epidemiology relies on accurate viral sequence data, so that small differences between similar viruses can be meaningfully interpreted. Despite its higher throughput and more detailed minority variant data, next-generation sequencing has yet to be widely adopted for HIV. The difficulty of accurately reconstructing the consensus sequence of a quasispecies from reads (short fragments of DNA) in the presence of large between- and within-host diversity, including frequent indels, may have presented a barrier. In particular, mapping (aligning) reads to a reference sequence leads to biased loss of information; this bias can distort epidemiological and evolutionary conclusions. De novo assembly avoids this bias by aligning the reads to themselves, producing a set of sequences called contigs. However contigs provide only a partial summary of the reads, misassembly may result in their having an incorrect structure, and no information is available at parts of the genome where contigs could not be assembled. To address these problems we developed the tool shiver to pre-process reads for quality and contamination, then map them to a reference tailored to the sample using corrected contigs supplemented with the user's choice of existing reference sequences. Run with two commands per sample, it can easily be used for large heterogeneous data sets. We used shiver to reconstruct the consensus sequence and minority variant information from paired-end short-read whole-genome data produced with the Illumina platform, for sixty-five existing publicly available samples and fifty new samples. We show the systematic superiority of mapping to shiver's constructed reference compared with mapping the same reads to the closest of 3,249 real references: median values of 13 bases called differently and more accurately, 0 bases called differently and less accurately, and 205 bases of missing sequence recovered. We also successfully applied sh
Langat P, Raghwani J, Dudas G, et al., 2017, Genome-wide evolutionary dynamics of influenza B viruses on a global scale., PLoS Pathogens, Vol: 13, ISSN: 1553-7366
The global-scale epidemiology and genome-wide evolutionary dynamics of influenza B remain poorly understood compared with influenza A viruses. We compiled a spatio-temporally comprehensive dataset of influenza B viruses, comprising over 2,500 genomes sampled worldwide between 1987 and 2015, including 382 newly-sequenced genomes that fill substantial gaps in previous molecular surveillance studies. Our contributed data increase the number of available influenza B virus genomes in Europe, Africa and Central Asia, improving the global context to study influenza B viruses. We reveal Yamagata-lineage diversity results from co-circulation of two antigenically-distinct groups that also segregate genetically across the entire genome, without evidence of intra-lineage reassortment. In contrast, Victoria-lineage diversity stems from geographic segregation of different genetic clades, with variability in the degree of geographic spread among clades. Differences between the lineages are reflected in their antigenic dynamics, as Yamagata-lineage viruses show alternating dominance between antigenic groups, while Victoria-lineage viruses show antigenic drift of a single lineage. Structural mapping of amino acid substitutions on trunk branches of influenza B gene phylogenies further supports these antigenic differences and highlights two potential mechanisms of adaptation for polymerase activity. Our study provides new insights into the epidemiological and molecular processes shaping influenza B virus evolution globally.
Fife MS, Whitehead T, Benkaroun J, et al., 2017, Gene knockout technology to characterise and ablate chicken Interferon Inducible Transmembrane Proteins (chIFITMs), 5th Annual Meeting of the International-Cytokine-and-Interferon-Society (ICIS), Publisher: ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, Pages: 74-75, ISSN: 1043-4666
Whitehead T, Steyn A, Benkaroun J, et al., 2017, Uncovering the role of chicken IFITM-mediated viral restriction, 5th Annual Meeting of the International-Cytokine-and-Interferon-Society (ICIS), Publisher: ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, Pages: 74-74, ISSN: 1043-4666
Sallah N, Carstensen T, Wakeham K, et al., 2017, Whole-genome association study of antibody response to Epstein-Barr virus in an African population: a pilot, Global Health, Epidemiology and Genomics, Vol: 2, ISSN: 2054-4200
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.
Scally SW, McLeod B, Bosch A, et al., 2017, Molecular definition of multiple sites of antibody inhibition of malaria transmission-blocking vaccine antigen Pfs25, Nature Communications, Vol: 8, ISSN: 2041-1723
The Plasmodium falciparum Pfs25 protein (Pfs25) is a leading malaria transmission-blocking vaccine antigen. Pfs25 vaccination is intended to elicit antibodies that inhibit parasite development when ingested by Anopheles mosquitoes during blood meals. The Pfs25 three-dimensional structure has remained elusive, hampering a molecular understanding of its function and limiting immunogen design. We report six crystal structures of Pfs25 in complex with antibodies elicited by immunization via Pfs25 virus-like particles in human immunoglobulin loci transgenic mice. Our structural findings reveal the fine specificities associated with two distinct immunogenic sites on Pfs25. Importantly, one of these sites broadly overlaps with the epitope of the well-known 4B7 mouse antibody, which can be targeted simultaneously by antibodies that target a non-overlapping site to additively increase parasite inhibition. Our molecular characterization of inhibitory antibodies informs on the natural disposition of Pfs25 on the surface of ookinetes and provides the structural blueprints to design next-generation immunogens.
McLaren PJ, Pulit SL, Gurdasani D, et al., 2017, Evaluating the impact of functional genetic variation on HIV-1 control, Journal of Infectious Diseases, Vol: 216, Pages: 1063-1069, ISSN: 0022-1899
BackgroundPrevious genetic association studies of human immunodeficiency virus-1 (HIV-1) progression have focused on common human genetic variation ascertained through genome-wide genotyping.MethodsWe sought to systematically assess the full spectrum of functional variation in protein coding gene regions on HIV-1 progression through exome sequencing of 1327 individuals. Genetic variants were tested individually and in aggregate across genes and gene sets for an influence on HIV-1 viral load.ResultsMultiple single variants within the major histocompatibility complex (MHC) region were observed to be strongly associated with HIV-1 outcome, consistent with the known impact of classical HLA alleles. However, no single variant or gene located outside of the MHC region was significantly associated with HIV progression. Set-based association testing focusing on genes identified as being essential for HIV replication in genome-wide small interfering RNA (siRNA) and clustered regularly interspaced short palindromic repeats (CRISPR) studies did not reveal any novel associations.ConclusionsThese results suggest that exonic variants with large effect sizes are unlikely to have a major contribution to host control of HIV infection.
Leitman EM, Thobakgale CF, Adland E, et al., 2017, Role of HIV-specific CD8(+) T cells in pediatric HIV cure strategies after widespread early viral escape, Journal of Experimental Medicine, Vol: 214, Pages: 3239-3261, ISSN: 0022-1007
Recent studies have suggested greater HIV cure potential among infected children than adults. A major obstacle to HIV eradication in adults is that the viral reservoir is largely comprised of HIV-specific cytotoxic T lymphocyte (CTL) escape variants. We here evaluate the potential for CTL in HIV-infected slow-progressor children to play an effective role in “shock-and-kill” cure strategies. Two distinct subgroups of children were identified on the basis of viral load. Unexpectedly, in both groups, as in adults, HIV-specific CTL drove the selection of escape variants across a range of epitopes within the first weeks of infection. However, in HIV-infected children, but not adults, de novo autologous variant-specific CTL responses were generated, enabling the pediatric immune system to “corner” the virus. Thus, even when escape variants are selected in early infection, the capacity in children to generate variant-specific anti-HIV CTL responses maintains the potential for CTL to contribute to effective shock-and-kill cure strategies in pediatric HIV infection.
Muir L, McKay P, Petrova V, et al., 2017, Optimisation of ex vivo memory B cell expansion/differentiation for interrogation of rare peripheral memory B cell subset responses [version 2; peer review: 2 approved], Wellcome Open Research, Vol: 2, ISSN: 2398-502X
Background: Human memory B cells play a vital role in the long-term protection of the host from pathogenic re-challenge. In recent years the importance of a number of different memory B cell subsets that can be formed in response to vaccination or infection has started to become clear. To study memory B cell responses, cells can be cultured ex vivo, allowing for an increase in cell number and activation of these quiescent cells, providing sufficient quantities of each memory subset to enable full investigation of functionality. However, despite numerous papers being published demonstrating bulk memory B cell culture, we could find no literature on optimised conditions for the study of memory B cell subsets, such as IgM+ memory B cells.Methods: Following a literature review, we carried out a large screen of memory B cell expansion conditions to identify the combination that induced the highest levels of memory B cell expansion. We subsequently used a novel Design of Experiments approach to finely tune the optimal memory B cell expansion and differentiation conditions for human memory B cell subsets. Finally, we characterised the resultant memory B cell subpopulations by IgH sequencing and flow cytometry.Results: The application of specific optimised conditions induce multiple rounds of memory B cell proliferation equally across Ig isotypes, differentiation of memory B cells to antibody secreting cells, and importantly do not alter the Ig genotype of the stimulated cells. Conclusions: Overall, our data identify a memory B cell culture system that offers a robust platform for investigating the functionality of rare memory B cell subsets to infection and/or vaccination.
Ba abdullah M, Palermo RD, Palser A, et al., 2017, Heterogeneity of the Epstein-Barr virus major internal repeat reveals evolutionary mechanisms of EBV and a functional defect in the prototype EBV strain B95-8., Journal of Virology, Vol: 91, ISSN: 1098-5514
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 both through co-evolution with its host, and genetic exchange between virus strains. Sequence analysis of the EBV genome is unusually challenging, because of the large number and length of repeat regions within the virus. Here we describe the sequence assembly and analysis of the large internal repeat of EBV (IR1 or BamW repeats) from over 70 strains.Diversity of the latency protein EBNA-LP resides predominantly within the exons downstream of IR1. The integrity of the putative BWRF1 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 spontaneous mutation, with inter-strain recombination more common in tumour-derived viruses. This genetic exchange often incorporates regions of <1kb, 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 non-consensus variants within a single IR1 repeat unit, including a STOP codon in EBNA-LP. Repairing IR1 improves EBNA-LP levels and the quality of transformation by the B95-8 BAC.
Thi Ty Hang V, Thi Han Ny N, My Phuc T, et al., 2017, Evaluation of the Luminex xTAG Respiratory Viral Panel FAST v2 assay for detection of multiple respiratory viral pathogens in nasal and throat swabs in Vietnam [version 1; referees: 2 approved], Wellcome Open Research, Vol: 2, ISSN: 2398-502X
Background: Acute respiratory infections (ARI) are among the leading causes of hospitalization in children ≤5 years old. Rapid diagnostics of viral pathogens is essential to avoid unnecessary antibiotic treatment, thereby slowing down antibiotic-resistance. We evaluated the diagnostic performance of the Luminex xTAG Respiratory Viral Panel FAST v2 against viral specific PCR as reference assays for ARI in Vietnam. Methods: Four hundred and forty two nose and throat swabs were collected in viral transport medium, and were tested with Luminex xTAG Respiratory Viral Panel FAST v2. Multiplex RT-PCR and single RT-PCR were used as references. Results: Overall, viral pathogens were detected in a total count of 270/294 (91.8%, 95% CI 88.1-94.7) by the Luminex among reference assays, whilst 112/6336 (1.8%, 95% CI, 1.4-2.1) of pathogens were detected by the Luminex, but not by reference assays. Frequency of pathogens detected by Luminex and reference assays was 379 and 292, respectively. The diagnostic yield was 66.7% (295/442, 95%CI 62.1-71.1%) for the Luminex assay and 54.1% (239/442, 95% CI, 49.3-58.8%) for reference assays. The Luminex kit had higher yields for all viruses except influenza B virus, respiratory syncytial virus, and human bocavirus. High agreements between both methods [mean (range): 0.91 (0.83-1.00)] were found for 10/15 viral agents. Conclusions: The Luminex assay is a high throughput multiplex platform for rapid detection of common viral pathogens causing ARI. Although the current high cost may prevent Luminex assays from being widely used, especially in limited resource settings where ARI are felt most, its introduction in clinical diagnostics may help reduce unnecessary use of antibiotic prescription.
Yebra G, Frampton D, Cassarino TG, et al., 2017, A High HIV-1 strain variability in London, UK, revealed by full-genome analysis: Results from the ICONIC project, Publisher: bioRxiv
Abstract Background & Methods The ICONIC project has developed an automated high-throughput pipeline to generate HIV nearly full-length genomes (NFLG, i.e. from gag to nef ) from next-generation sequencing (NGS) data. The pipeline was applied to 420 HIV samples collected at University College London Hospital and Barts Health NHS Trust (London) and sequenced using an Illumina MiSeq at the Wellcome Trust Sanger Institute (Cambridge). Consensus genomes were generated and subtyped using COMET, and unique recombinants were studied with jpHMM and SimPlot. Maximum-likelihood phylogenetic trees were constructed using RAxML to identify transmission networks using the Cluster Picker. Results The pipeline generated sequences of at least 1Kb of length (median=7.4Kb) for 375 out of the 420 samples (89%), with 174 (46.4%) being NFLG. A total of 365 sequences (169 of them NFLG) corresponded to unique subjects and were included in the down-stream analyses. The most frequent HIV subtypes were B (n=149, 40.8%) and C (n=77, 21.1%) and the circulating recombinant form CRF02_AG (n=32, 8.8%). We found 14 different CRFs (n=66, 18.1%) and multiple URFs (n=32, 8.8%) that involved recombination between 12 different subtypes/CRFs. The most frequent URFs were B/CRF01_AE (4 cases) and A1/D, B/C, and B/CRF02_AG (3 cases each). Most URFs (19/26, 73%) lacked breakpoints in the PR+RT pol region, rendering them undetectable if only that was sequenced. Twelve (37.5%) of the URFs could have emerged within the UK, whereas the rest were probably imported from sub-Saharan Africa, South East Asia and South America. For 2 URFs we found highly similar pol sequences circulating in the UK. We detected 31 phylogenetic clusters using the full dataset: 25 pairs (mostly subtypes B and C), 4 triplets and 2 quadruplets. Some of these were not consistent across different genes due to inter- and intra-subtype recombination. Clusters involved 70 sequences, 19.2% of the dataset. Conclusions The initial analysis of
Correia S, Palser A, Elgueta Karstegl C, et al., 2017, Natural variation of Epstein-Barr virus genes, proteins and pri-microRNA, Journal of Virology, Vol: 91, ISSN: 1098-5514
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 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 SNPs in the pri-miRNA outside of 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 variation with EBV biology and EBV associated diseases.IMPORTANCE Incidence of diseases associated with EBV varies greatly in different parts of the world. Relationships between EBV genome sequence variation and health, disease, geography and ethnicity of the host may thus 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 focussing on variation in LMP1, Zp, gp350, EBNA1 and the BART miRNA cluster 2, new relationships to the known type 1/type 2 strains are demonstrated and novel classificati
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