Daniel P. Depledge

Specific Capture and Whole-Genome Sequencing of Viruses from Clinical Samples

Whole genome sequencing of viruses directly from clinical samples is integral for understanding the genetics of host-virus interactions. Here, we report the use of sample sparing target enrichment (by hybridisation) for viral nucleic acid separation and deep-sequencing of herpesvirus genomes directly from a range of clinical samples including saliva, blood, virus vesicles, cerebrospinal fluid, and tumour cell lines. We demonstrate the effectiveness of the method by deep-sequencing 13 highly cell-associated human herpesvirus genomes and generating full length genome alignments at high read depth. Moreover, we show the specificity of the method enables the study of viral population structures and their diversity within a range of clinical samples types.

Viral population analysis and minority-variant detection using short read next-generation sequencing

RNA viruses within infected individuals exist as a population of evolutionary-related variants. Owing to evolutionary change affecting the constitution of this population, the frequency and/or occurrence of individual viral variants can show marked or subtle fluctuations. Since the development of massively parallel sequencing platforms, such viral populations can now be investigated to unprecedented resolution. A critical problem with such analyses is the presence of sequencing-related errors that obscure the identification of true biological variants present at low frequency. Here, we report the development and assessment of the Quality Assessment of Short Read (QUASR) Pipeline (http://sourceforge.net/projects/quasr) specific for virus genome short read analysis that minimizes sequencing errors from multiple deep-sequencing platforms, and enables post-mapping analysis of the minority variants within the viral population. QUASR significantly reduces the error-related noise in deep-sequencing datasets, resulting in increased mapping accuracy and reduction of erroneous mutations. Using QUASR, we have determined influenza virus genome dynamics in sequential samples from an in vitro evolution of 2009 pandemic H1N1 (A/H1N1/09) influenza from samples sequenced on both the Roche 454 GSFLX and Illumina GAIIx platforms. Importantly, concordance between the 454 and Illumina sequencing allowed unambiguous minority-variant detection and accurate determination of virus population turnover in vitro.

Rapid Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates Directly from Clinical Samples.

ABSTRACT The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures of M. tuberculosis. Here we describe a method utilizing biotinylated RNA baits designed specifically for M. tuberculosis DNA to capture full M. tuberculosis genomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the United Kingdom and Lithuania where a matched culture sample was available, and 2 samples that had failed to grow in culture. M. tuberculosis sequencing data were obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were of high quality (>20× depth and >90% of the genome covered). Results were compared with those of conventional molecular and culture-based methods, and high levels of concordance between phenotypical resistance and predicted resistance based on genotype were observed. High-quality sequence data were obtained from one smear-positive culture-negative case. This study demonstrated for the first time the successful and accurate sequencing of M. tuberculosis genomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalized rather than empirical treatment of drug-resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes.

Next-Generation Whole Genome Sequencing Identifies the Direction of Norovirus Transmission in Linked Patients

BACKGROUND Noroviruses are a highly transmissible and major cause of nosocomial gastroenteritis resulting in bed and hospital-ward closures. Where hospital outbreaks are suspected, it is important to determine the routes of spread so that appropriate infection-control procedures can be implemented. To investigate a cluster of norovirus cases occurring in children undergoing bone marrow transplant, we undertook norovirus genome sequencing by next-generation methods. Detailed comparison of sequence data from 2 linked cases enabled us to identify the likely direction of spread. METHODS Norovirus complementary DNA was amplified by overlapping polymerase chain reaction (PCR) from 13 stool samples from 5 diagnostic real-time PCR-positive patients. The amplicons were sequenced by Roche 454, the genomes assembled by de novo assembly, and the data analyzed phylogenetically. RESULTS Phylogenetic analysis indicated that patients were infected by viruses similar to 4 distinct GII.4 subtypes and 2 patients were linked by the same virus. Of the 14 sites at which there were differences between the consensus sequences of the 2 linked viral genomes, 9 had minor variants present within one or the other patient. Further analysis confirmed that minor variants at all 9 sites in patient B w ere present as the consensus sequence in patient A. CONCLUSIONS Phylogenetic analysis excluded a common source of infection in this apparent outbreak. Two of 3 patients on the same ward had closely related viruses, raising the possibility of cross-infection despite protective isolation. Analysis of deep sequencing data enabled us to establish the likely direction of nosocomial transmission.

Deep sequencing of viral genomes provides insight into the evolution and pathogenesis of varicella zoster virus and its vaccine in humans

Immunization with the vOka vaccine prevents varicella (chickenpox) in children and susceptible adults. The vOka vaccine strain comprises a mixture of genotypes and, despite attenuation, causes rashes in small numbers of recipients. Like wild-type virus, the vaccine establishes latency in neuronal tissue and can later reactivate to cause Herpes zoster (shingles). Using hybridization-based methodologies, we have purified and sequenced vOka directly from skin lesions. We show that alleles present in the vaccine can be recovered from the lesions and demonstrate the presence of a severe bottleneck between inoculation and lesion formation. Genotypes in any one lesion appear to be descended from one to three vaccine-genotypes with a low frequency of novel mutations. No single vOka haplotype and no novel mutations are consistently present in rashes, indicating that neither new mutations nor recombination with wild type are critical to the evolution of vOka rashes. Instead, alleles arising from attenuation (i.e., not derived from free-living virus) are present at lower frequencies in rash genotypes. We identify 11 loci at which the ancestral allele is selected for in vOka rash formation and show genotypes in rashes that have reactivated from latency cannot be distinguished from rashes occurring immediately after inoculation. We conclude that the vOka vaccine, although heterogeneous, has not evolved to form rashes through positive selection in the mode of a quasispecies, but rather alleles that were essentially neutral during the vaccine production have been selected against in the human subjects, allowing us to identify key loci for rash formation.

Recombination of Globally Circulating Varicella Zoster Virus

ABSTRACT Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpesvirus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine-wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo or in vitro during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade. IMPORTANCE Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the present study, we demonstrate that VZV also frequently undergoes genetic recombination, including strains belonging to the clade containing the vOKA strain.

Whole-genome enrichment and sequencing of Chlamydia trachomatis directly from clinical samples

BackgroundChlamydia trachomatis is a pathogen of worldwide importance, causing more than 100 million cases of sexually transmitted infections annually. Whole-genome sequencing is a powerful high resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography and mutations associated with antimicrobial resistance. The objective of this study was to perform whole-genome enrichment and sequencing of C. trachomatis directly from clinical samples.MethodsC. trachomatis positive samples comprising seven vaginal swabs and three urine samples were sequenced without prior in vitro culture in addition to nine cultured C. trachomatis samples, representing different serovars. A custom capture RNA bait set, that captures all known diversity amongst C. trachomatis genomes, was used in a whole-genome enrichment step during library preparation to enrich for C. trachomatis DNA. All samples were sequenced on the MiSeq platform.ResultsFull length C. trachomatis genomes (>95-100% coverage of a reference genome) were successfully generated for eight of ten clinical samples and for all cultured samples. The proportion of reads mapping to C. trachomatis and the mean read depth across each genome were strongly linked to the number of bacterial copies within the original sample. Phylogenetic analysis confirmed the known population structure and the data showed potential for identification of minority variants and mutations associated with antimicrobial resistance. The sensitivity of the method was >10-fold higher than other reported methodologies.ConclusionsThe combination of whole-genome enrichment and deep sequencing has proven to be a non-mutagenic approach, capturing all known variation found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.

In vitro system using human neurons demonstrates that varicella-zoster vaccine virus is impaired for reactivation, but not latency

Significance The varicella vaccine is highly effective in preventing chickenpox. Although the vaccine virus is attenuated, it is unknown whether the virus is impaired for establishment of latency, for reactivation, or both. We developed an in vitro system using human neurons derived from ES cells and showed that axonal infection of neurons results in latent infection and that virus can be reactivated using Ab to NGF. Using this system, we show that the varicella vaccine is impaired for reactivation, but not latency. Although not necessarily equivalent to varicella-zoster virus (VZV) latency and reactivation in vivo, this system may be used to study the ability of other viruses, including viral vaccines, to establish latency and reactivate. Varicella-zoster virus (VZV) establishes latency in human sensory and cranial nerve ganglia during primary infection (varicella), and the virus can reactivate and cause zoster after primary infection. The mechanism of how the virus establishes and maintains latency and how it reactivates is poorly understood, largely due to the lack of robust models. We found that axonal infection of neurons derived from hESCs in a microfluidic device with cell-free parental Oka (POka) VZV resulted in latent infection with inability to detect several viral mRNAs by reverse transcriptase-quantitative PCR, no production of infectious virus, and maintenance of the viral DNA genome in endless configuration, consistent with an episome configuration. With deep sequencing, however, multiple viral mRNAs were detected. Treatment of the latently infected neurons with Ab to NGF resulted in production of infectious virus in about 25% of the latently infected cultures. Axonal infection of neurons with vaccine Oka (VOka) VZV resulted in a latent infection similar to infection with POka; however, in contrast to POka, VOka-infected neurons were markedly impaired for reactivation after treatment with Ab to NGF. In addition, viral transcription was markedly reduced in neurons latently infected with VOka compared with POka. Our in vitro system recapitulates both VZV latency and reactivation in vivo and may be used to study viral vaccines for their ability to establish latency and reactivate.

Rates of vaccine evolution show strong effects of latency: implications for Varicella Zoster virus epidemiology.

Varicella-zoster virus (VZV) causes chickenpox and shingles, and is found in human populations worldwide. The lack of temporal signal in the diversity of VZV makes substitution rate estimates unreliable, which is a barrier to understanding the context of its global spread. Here, we estimate rates of evolution by studying live attenuated vaccines, which evolved in 22 vaccinated patients for known periods of time, sometimes, but not always undergoing latency. We show that the attenuated virus evolves rapidly (∼10−6 substitutions/site/day), but that rates decrease dramatically when the virus undergoes latency. These data are best explained by a model in which viral populations evolve for around 13 days before becoming latent, but then undergo no replication during latency. This implies that rates of viral evolution will depend strongly on transmission patterns. Nevertheless, we show that implausibly long latency periods are required to date the most recent common ancestor of extant VZV to an “out-of-Africa” migration with humans, as has been previously suggested.

Evolution of Cocirculating Varicella-Zoster Virus Genotypes during a Chickenpox Outbreak in Guinea-Bissau

ABSTRACT Varicella-zoster virus (VZV), a double-stranded DNA alphaherpesvirus, is associated with seasonal outbreaks of varicella in nonimmunized populations. Little is known about whether these outbreaks are associated with a single or multiple viral genotypes and whether new mutations rapidly accumulate during transmission. Here, we take advantage of a well-characterized population cohort in Guinea-Bissau and produce a unique set of 23 full-length genome sequences, collected over 7 months from eight households. Comparative sequence analysis reveals that four distinct genotypes cocirculated among the population, three of which were present during the first week of the outbreak, although no patients were coinfected, which indicates that exposure to infectious virus from multiple sources is common during VZV outbreaks. Transmission of VZV was associated with length polymorphisms in the R1 repeat region and the origin of DNA replication. In two cases, these were associated with the formation of distinct lineages and point to the possible coevolution of these loci, despite the lack of any known functional link in VZV or related herpesviruses. We show that these and all other sequenced clade 5 viruses possess a distinct R1 repeat motif that increases the acidity of an ORF11p protein domain and postulate that this has either arisen or been lost following divergence of the major clades. Thus, sequencing of whole VZV genomes collected during an outbreak has provided novel insights into VZV biology, transmission patterns, and (recent) natural history. IMPORTANCE VZV is a highly infectious virus and the causative agent of chickenpox and shingles, the latter being particularly associated with the risk of painful complications. Seasonal outbreaks of chickenpox are very common among young children, yet little is known about the dynamics of the virus during person-to-person to transmission or whether multiple distinct viruses seed and/or cocirculate during an outbreak. In this study, we have sequenced chickenpox viruses from an outbreak in Guinea-Bissau that are supported by detailed epidemiological data. Our data show that multiple different virus strains seeded and were maintained throughout the 6-month outbreak period and that viruses transmitted between individuals accumulated new mutations in specific genomic regions. Of particular interest is the potential coevolution of two distinct parts of the genomes and our calculations of the rate of viral mutation, both of which increase our understanding of how VZV evolves over short periods of time in human populations.