Astrid Gall

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

Summary 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.

Disease-associated XMRV sequences are consistent with laboratory contamination

BackgroundXenotropic murine leukaemia viruses (MLV-X) are endogenous gammaretroviruses that infect cells from many species, including humans. Xenotropic murine leukaemia virus-related virus (XMRV) is a retrovirus that has been the subject of intense debate since its detection in samples from humans with prostate cancer (PC) and chronic fatigue syndrome (CFS). Controversy has arisen from the failure of some studies to detect XMRV in PC or CFS patients and from inconsistent detection of XMRV in healthy controls.ResultsHere we demonstrate that Taqman PCR primers previously described as XMRV-specific can amplify common murine endogenous viral sequences from mouse suggesting that mouse DNA can contaminate patient samples and confound specific XMRV detection. To consider the provenance of XMRV we sequenced XMRV from the cell line 22Rv1, which is infected with an MLV-X that is indistinguishable from patient derived XMRV. Bayesian phylogenies clearly show that XMRV sequences reportedly derived from unlinked patients form a monophyletic clade with interspersed 22Rv1 clones (posterior probability >0.99). The cell line-derived sequences are ancestral to the patient-derived sequences (posterior probability >0.99). Furthermore, pol sequences apparently amplified from PC patient material (VP29 and VP184) are recombinants of XMRV and Moloney MLV (MoMLV) a virus with an envelope that lacks tropism for human cells. Considering the diversity of XMRV we show that the mean pairwise genetic distance among env and pol 22Rv1-derived sequences exceeds that of patient-associated sequences (Wilcoxon rank sum test: p = 0.005 and p < 0.001 for pol and env, respectively). Thus XMRV sequences acquire diversity in a cell line but not in patient samples. These observations are difficult to reconcile with the hypothesis that published XMRV sequences are related by a process of infectious transmission.ConclusionsWe provide several independent lines of evidence that XMRV detected by sensitive PCR methods in patient samples is the likely result of PCR contamination with mouse DNA and that the described clones of XMRV arose from the tumour cell line 22Rv1, which was probably infected with XMRV during xenografting in mice. We propose that XMRV might not be a genuine human pathogen.

Design and validation of a microarray for detection, hemagglutinin subtyping, and pathotyping of avian influenza viruses.

ABSTRACT Continuing threats of devastating outbreaks in poultry and of human infections caused by highly pathogenic avian influenza virus (HPAIV) H5N1 emphasize the need for the further development of rapid and reliable methods of virus detection and characterization. Here we report on the design and comprehensive validation of a low-density microarray as a diagnostic tool for the detection and typing of avian influenza virus (AIV). The array consists of one probe for the conserved matrix gene and 97 probes targeting the HA0 cleavage-site region. Following fragment amplification by a generic PCR approach, the array enables AIV detection, hemagglutinin (HA) subtyping, and pathotyping within a single assay. For validation, a panel of 92 influenza A viruses which included 43 reference strains representing all 16 HA subtypes was used. All reference strains were correctly typed with respect to their HA subtypes and pathotypes, including HPAIV H5N1/Asia, which caused outbreaks in Germany in 2006 and 2007. In addition, differentiation of strains of the Eurasian and North American lineages of the H5 and H7 subtypes was possible. The sensitivity of the microarray for the matrix gene is comparable to that of real-time reverse transcription-PCR (RT-PCR). It is, however, 10- to 100-fold lower than that of real-time RT-PCR with respect to HA subtyping and pathotyping. The specificity of the array was excellent, as no pathogens relevant for differential diagnosis yielded a positive reaction. Validation with field samples included 19 cloacal swab specimens from wild and domestic birds. Influenza A virus was verified in all samples, whereas the HA subtypes could be determined for 14 samples. The results demonstrate that the microarray assay described complements current methods and can accelerate the diagnosis and characterization of AIV.

Universal Primer Set for Amplification and Sequencing of HA0 Cleavage Sites of All Influenza A Viruses

ABSTRACT Sequence analysis of the endoproteolytic cleavage site within the hemagglutinin (HA) precursor protein HA0 is fundamental for studies of the molecular biology of influenza A viruses, in particular, for molecular pathotyping of subtype H5 and H7 isolates. A current problem for routine diagnostics is the emergence of new strains of the H5 or H7 subtype or even other subtypes which escape detection by commonly used reverse transcription-PCR (RT-PCR) protocols. Here, the first pan-HA (PanHA) RT-PCR assay targeting the HA0 cleavage site of influenza A viruses of all 16 HA subtypes is reported. The assay was assessed in comparison to H5 and H7 subtype-specific RT-PCRs for the HA0 cleavage site and a real-time RT-PCR detecting the M gene. A panel of 92 influenza A viruses was used for validation. Sequence data for influenza A viruses from 32 allantoic fluid samples and 11 diagnostic swab samples of all 16 HA subtypes were generated by direct sequencing of the PanHA RT-PCR products. The results demonstrate that the new PanHA RT-PCR assay—followed by cycle sequencing—can complement existing methods and strengthen the reliability of influenza A virus diagnostics, allowing both molecular pathotyping (H5 and H7) and subtyping (non-H5 or -H7) within a single approach.

IVA: accurate de novo assembly of RNA virus genomes

Motivation: An accurate genome assembly from short read sequencing data is critical for downstream analysis, for example allowing investigation of variants within a sequenced population. However, assembling sequencing data from virus samples, especially RNA viruses, into a genome sequence is challenging due to the combination of viral population diversity and extremely uneven read depth caused by amplification bias in the inevitable reverse transcription and polymerase chain reaction amplification process of current methods. Results: We developed a new de novo assembler called IVA (Iterative Virus Assembler) designed specifically for read pairs sequenced at highly variable depth from RNA virus samples. We tested IVA on datasets from 140 sequenced samples from human immunodeficiency virus-1 or influenza-virus-infected people and demonstrated that IVA outperforms all other virus de novo assemblers. Availability and implementation: The software runs under Linux, has the GPLv3 licence and is freely available from http://sanger-pathogens.github.io/iva Contact: iva@sanger.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Rapid haemagglutinin subtyping and pathotyping of avian influenza viruses by a DNA microarray

Rapid and reliable methods are fundamental for the comprehensive characterization of emerging and evolving avian influenza viruses. Although microarrays provide new possibilities with their parallel approach, their use in diagnostic laboratories is still limited due to economical and practical factors. An easy-to-use, low-cost microarray-based assay for haemagglutinin subtyping and pathotyping of avian influenza viruses and specific detection of highly pathogenic H5N1/Asia clade 2.2 is described as a novel diagnostic tool. The ArrayTube platform is user-friendly, inexpensive and allows processing of many samples. The sensitivity of the assay developed was comparable to real-time RT-PCR, and the simultaneous detection of different subtypes was possible. Validation with 90 influenza A virus isolates representing all 16 haemagglutinin subtypes and 44 field samples (cloacal swabs from wild and domestic birds) demonstrated the feasibility of the system for sensitive and specific characterization of AIV. Facilitating haemagglutinin subtyping and pathotyping for the majority of influenza A-positive cloacal swabs within 24h, the new assay enables detailed AIV diagnosis even in less well-equipped laboratories.

Rapid and Highly Sensitive Neuraminidase Subtyping of Avian Influenza Viruses by Use of a Diagnostic DNA Microarray

ABSTRACT Rapid neuraminidase subtyping of avian influenza viruses from diagnostic samples is crucial considering the existence of permanently emerging and evolving strains. Here we report an easy-to-use, low-cost microarray for neuraminidase subtyping following fragment amplification by a generic, neuraminidase-specific reverse transcription-PCR (RT-PCR). This method enables highly specific characterization with a sensitivity equal to that of matrix gene-specific real-time RT-PCR.

Restriction of V3 region sequence divergence in the HIV-1 envelope gene during antiretroviral treatment in a cohort of recent seroconverters.

BackgroundDynamic changes in Human Immunodeficiency Virus 1 (HIV-1) sequence diversity and divergence are associated with immune control during primary infection and progression to AIDS. Consensus sequencing or single genome amplification sequencing of the HIV-1 envelope (env) gene, in particular the variable (V) regions, is used as a marker for HIV-1 genome diversity, but population diversity is only minimally, or semi-quantitatively sampled using these methods.ResultsHere we use second generation deep sequencing to determine inter-and intra-patient sequence heterogeneity and to quantify minor variants in a cohort of individuals either receiving or not receiving antiretroviral treatment following seroconversion; the SPARTAC trial. We show, through a cross-sectional study of sequence diversity of the env V3 in 30 antiretroviral-naive patients during primary infection that considerable population structure diversity exists, with some individuals exhibiting highly constrained plasma virus diversity. Diversity was independent of clinical markers (viral load, time from seroconversion, CD4 cell count) of infection. Serial sampling over 60 weeks of non-treated individuals that define three initially different diversity profiles showed that complex patterns of continuing HIV-1 sequence diversification and divergence could be readily detected. Evidence for minor sequence turnover, emergence of new variants and re-emergence of archived variants could be inferred from this analysis. Analysis of viral divergence over the same time period in patients who received short (12 weeks, ART12) or long course antiretroviral therapy (48 weeks, ART48) and a non-treated control group revealed that ART48 successfully suppressed viral divergence while ART12 did not have a significant effect.ConclusionsDeep sequencing is a sensitive and reliable method for investigating the diversity of the env V3 as an important component of HIV-1 genome diversity. Detailed insights into the complex early intra-patient dynamics of env V3 diversity and divergence were explored in antiretroviral-naïve recent seroconverters. Long course antiretroviral therapy, initiated soon after seroconversion and administered for 48 weeks, restricts HIV-1 divergence significantly. The effect of ART12 and ART48 on clinical markers of HIV infection and progression is currently investigated in the SPARTAC trial.

Viral genetic variation accounts for a third of variability in HIV-1 set-point viral load in Europe.

HIV-1 set-point viral load—the approximately stable value of viraemia in the first years of chronic infection—is a strong predictor of clinical outcome and is highly variable across infected individuals. To better understand HIV-1 pathogenesis and the evolution of the viral population, we must quantify the heritability of set-point viral load, which is the fraction of variation in this phenotype attributable to viral genetic variation. However, current estimates of heritability vary widely, from 6% to 59%. Here we used a dataset of 2,028 seroconverters infected between 1985 and 2013 from 5 European countries (Belgium, Switzerland, France, the Netherlands and the United Kingdom) and estimated the heritability of set-point viral load at 31% (CI 15%–43%). Specifically, heritability was measured using models of character evolution describing how viral load evolves on the phylogeny of whole-genome viral sequences. In contrast to previous studies, (i) we measured viral loads using standardized assays on a sample collected in a strict time window of 6 to 24 months after infection, from which the viral genome was also sequenced; (ii) we compared 2 models of character evolution, the classical “Brownian motion” model and another model (“Ornstein–Uhlenbeck”) that includes stabilising selection on viral load; (iii) we controlled for covariates, including age and sex, which may inflate estimates of heritability; and (iv) we developed a goodness of fit test based on the correlation of viral loads in cherries of the phylogenetic tree, showing that both models of character evolution fit the data well. An overall heritability of 31% (CI 15%–43%) is consistent with other studies based on regression of viral load in donor–recipient pairs. Thus, about a third of variation in HIV-1 virulence is attributable to viral genetic variation.

Dynamics of immunoglobulin sequence diversity in HIV-1 infected individuals

Advances in immunoglobulin (Ig) sequencing technology are leading to new perspectives on immune system dynamics. Much research in this nascent field has focused on resolving immune responses to viral infection. However, the dynamics of B-cell diversity in early HIV infection, and in response to anti-retroviral therapy, are still poorly understood. Here, we investigate these dynamics through bulk Ig sequencing of samples collected over 2 years from a group of eight HIV-1 infected patients, five of whom received anti-retroviral therapy during the first half of the study period. We applied previously published methods for visualizing and quantifying B-cell sequence diversity, including the Gini index, and compared their efficacy to alternative measures. While we found significantly greater clonal structure in HIV-infected patients versus healthy controls, within HIV patients, we observed no significant relationships between statistics of B-cell clonal expansion and clinical variables such as viral load and CD4+ count. Although there are many potential explanations for this, we suggest that important factors include poor sampling resolution and complex B-cell dynamics that are difficult to summarize using simple summary statistics. Importantly, we find a significant association between observed Gini indices and sequencing read depth, and we conclude that more robust analytical methods and a closer integration of experimental and theoretical work is needed to further our understanding of B-cell repertoire diversity during viral infection.