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Background: Many patients with non-small cell lung cancer (NSCLC) lack access to highly effective approved targeted therapeutics due to multiple gaps in biomarker testing. Challenges in comprehensive molecular testing include complexities associated with the need to assess the presence of multiple variants, costs of running multiple sequential assays per sample, high assay quality control (QC) failure rates, clinical need for rapid turn-around time (TAT) to initiate therapy, and insufficient tissue samples. The ASPYRE-Lung NSCLC assay addresses gaps in multiplexed testing by simultaneously analyzing DNA and RNA, detecting 114 actionable genomic variants across 11 genes, consistent with current NSCLC treatment guidelines. This study was to assess the ease of adoption and performance of ASPYRE-Lung in third-party laboratories, comparing concordance across sites and with orthogonal methods., Methods: ASPYRE-Lung was established at two academic centers with multiple operators per site. Assay concordance was evaluated across three sites using 77 patient samples [61 derived from formalin-fixed paraffin-embedded (FFPE) tissue and 16 from cytology specimens]., Results: Reproducibility for all 77 samples yielded a positive percent agreement (PPA) of 100% and negative percent agreement (NPA) of 99.99%. Concordance with next-generation sequencing (NGS)-based methods across all three sites was high with PPA of 97.2% and NPA of 99.96%., Conclusions: ASPYRE-Lung assay is a cost-effective, easy to adopt testing method requiring no specialized expertise or complicated bioinformatics, with the potential to inform genomic data on small tissue samples, thus enabling all patients with NSCLC to undergo biomarker testing in a timely manner and benefit from appropriate targeted therapies., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-24-525/coif). All authors report that this work was supported by Biofidelity Ltd., UK. S.E.H. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. She served as technical consultant for potential investors at Biofidelity (Oct 2023 to Jan 2024). She received honoraria and travel expenses from ThermoFisher to present on NSCLC at a corporate workshop at AMP 2024. C.G. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. S.J.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. B.A.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. K.A.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. K.F.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. J.M.M. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. J.M.M. is an employee of Biofidelity Ltd. and has financial interest including salary, stocks, and stock options. R.N.P. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. R.N.P. is an employee of Biofidelity Ltd. and has financial interest including salary, stocks, and stock options. C.X. reports that Biofidelity provided all materials (reagents, consumables, travel expenses to laboratories) to complete the work. C.X. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. E.G.Z. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. E.G.Z. is an employee of Biofidelity Inc. and has financial interest including salary, stock, and stock options. C.K. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. C.K. is an employee of Biofidelity Inc. and has financial interest including salary, stock, and stock options. R.T.E. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. R.T.E. is an employee of Biofidelity Inc. and has financial interest including salary, stock and stock options. A.S.G. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. A.S.G. is an employee of Biofidelity Inc. and has financial interest including salary, stock, and stock options. A.L.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. A.L.S. is an employee of Biofidelity Inc. or Biofidelity Ltd. and has financial interest including salary, intellectual property, stock, and stock options. M.S.J. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. M.S.J. is an employee of Biofidelity Ltd. and has financial interest including salary, intellectual property, stock, and stock options. K.v.B. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. K.v.B. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. I.T. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. I.T. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. C.H.H. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. C.H.H. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. A. Collazos reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. A. Collazos is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. N.D.P. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. N.D.P. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. D.N. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. D.N. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. J.J. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. J.J. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. E.R.G. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. E.R.G. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. E.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. E.S. is an employee of Biofidelity Inc. and has financial interest including salary, stock, and stock options. E.S. is a former employee of Adaptive Biotechnologies, which have no interests in the work presented herein. W.J.L. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. WJL. is a consultant of Biofidelity Inc. and has financial interest including consultancy fees and work-related travel to AACR 2024 where data from this work was presented. A. Cooke reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. A. Cooke was an employee of Biofidelity Ltd. and has financial interest including salary and stock. B.W.B. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. B.W.B. reports that Biofidelity Ltd. has provided travel expenses for work-related travel to AACR 2024 where data from this work was presented. B.W.B. is an employee of Biofidelity Ltd. and has financial interest including salary, intellectual property, stock, and stock options. R.J.O. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. R.J.O. was an employee of Biofidelity Ltd., and has financial interest including salary, stock, and stock options. H.V.R. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. H.V.R. was a consultant of Biofidelity Inc. and had financial interest including consultancy fees. H.V.R. is a current employee of Belay Diagnostics, which have no interests in the work presented herein. V.M.V.D. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. The authors have no other conflicts of interest to declare., (2024 AME Publishing Company. All rights reserved.)

Introduction: Genomic variant testing of tumors is a critical gateway for patients to access the full potential of personalized oncology therapeutics. Current methods such as next-generation sequencing are costly and challenging to interpret, while PCR assays are limited in the number of variants they can cover. We developed ASPYRE ® (Allele-Specific PYrophosphorolysis REaction) technology to address the urgent need for rapid, accessible and affordable diagnostics informing actionable genomic target variants of a given cancer. The targeted ASPYRE-Lung panel for non-small cell carcinoma covers 114 variants in 11 genes ( ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET & NTRK1/2/3 ) to robustly inform clinical management. The assay detects single nucleotide variants, insertions, deletions, and gene fusions from tissue-derived DNA and RNA simultaneously., Methods: We tested the limit of detection, specificity, analytical accuracy and analytical precision of ASPYRE-Lung using FFPE lung tissue samples from patients with non-small cell lung carcinoma, variant-negative FFPE tissue from healthy donors, and FFPE-based contrived samples with controllable variant allele fractions., Results: The sensitivity of ASPYRE-Lung was determined to be ≤ 3% variant allele fraction for single nucleotide variants and insertions or deletions, 100 copies for fusions, and 200 copies for MET exon 14 skipping. The specificity was 100% with no false positive results. The analytical accuracy test yielded no discordant calls between ASPYRE-Lung and expected results for clinical samples (via orthogonal testing) or contrived samples, and results were replicable across operators, reagent lots, runs, and real-time PCR instruments with a high degree of precision., Conclusions: The technology is simple and fast, requiring only four reagent transfer steps using standard laboratory equipment (PCR and qPCR instruments) with analysis via a cloud-based algorithm. The ASPYRE-Lung assay has the potential to be transformative in facilitating access to rapid, actionable molecular profiling of tissue for patients with non-small cell carcinoma., Competing Interests: Authors RE, EG-Z, JB, KK, CK, AG, and BB are employed by the company Biofidelity Inc. Authors A-LS, JM, RP, AT, AC, CX, IT, CH, DN, JJ, SA, NP, KB, EG, MS-J, AC and RO are employees of Biofidelity Ltd, a privately held company. All authors may hold stock or stock options. Author HR was a consultant of Biofidelity Inc at the time of the study. Biofidelity has filed patent applications on aspects of this research WO2021130494A1. The author HR declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision. The authors declare that this study received funding from Biofidelity Ltd. The following had the following involvement in the study: study design, data collection, analysis, decision to publish, and preparation of the manuscript., (Copyright © 2024 Evans, Gillon-Zhang, Brown, Knudsen, King, Green, Silva, Mordaka, Palmer, Tomassini, Collazos, Xyrafaki, Turner, Ho, Nugent, Jose, Andreazza, Potts, von Bargen, Gray, Stolarek-Januszkiewicz, Cooke, Reddi, Balmforth and Osborne.)

Protein-protein interactions are fundamental to life processes. Complementary computational, structural, and biophysical studies of these interactions enable the forces behind their specificity and strength to be understood. Antibody fragments such as single-chain antibodies have the specificity and affinity of full antibodies but a fraction of their size, expediting whole molecule studies and distal effects without exceeding the computational capacity of modeling systems. We previously reported the crystal structure of a high-affinity nanobody 59H10 bound to HIV-1 capsid protein p24 and deduced key interactions using all-atom molecular dynamics simulations. We studied the properties of closely related medium (37E7) and low (48G11) affinity nanobodies, to understand how changes of three (37E7) or one (48G11) amino acids impacted these interactions; however, the contributions of enthalpy and entropy were not quantified. Here, we report the use of qualitative and quantitative experimental and in silico approaches to separate the contributions of enthalpy and entropy. We used complementary circular dichroism spectroscopy and molecular dynamics simulations to qualitatively delineate changes between nanobodies in isolation and complexed with p24. Using quantitative techniques such as isothermal titration calorimetry alongside WaterMap and Free Energy Perturbation protocols, we found the difference between high (59H10) and medium (37E7) affinity nanobodies on binding to HIV-1 p24 is entropically driven, accounted for by the release of unstable waters from the hydrophobic surface of 59H10. Our results provide an exemplar of the utility of parallel in vitro and in silico studies and highlight that differences in entropic interactions between amino acids and water molecules are sufficient to drive orders of magnitude differences in affinity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background: RNA is a critical analyte for unambiguous detection of actionable mutations used to guide treatment decisions in oncology. Currently available methods for gene fusion detection include molecular or antibody-based assays, which suffer from either being limited to single-gene targeting, lack of sensitivity, or long turnaround time. The sensitivity and predictive value of next generation sequencing DNA-based assays to detect fusions by sequencing intronic regions is variable, due to the extensive size of introns. The required depth of sequencing and input nucleic acid required can be prohibitive; in addition it is not certain that predicted gene fusions are actually expressed., Results: Herein we describe a method based on pyrophosphorolysis to include detection of gene fusions from RNA, with identical assay steps and conditions to detect somatic mutations in DNA [1], permitting concurrent assessment of DNA and RNA in a single instrument run., Conclusion: The limit of detection was under 6 molecules/ 6 µL target volume. The workflow and instrumentation required are akin to PCR assays, and the entire assay from extracted nucleic acid to sample analysis can be completed within a single day., (© 2022. The Author(s).)

Accurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

The quantum spin properties of nitrogen-vacancy defects in diamond enable diverse applications in quantum computing and communications 1 . However, fluorescent nanodiamonds also have attractive properties for in vitro biosensing, including brightness 2 , low cost 3 and selective manipulation of their emission 4 . Nanoparticle-based biosensors are essential for the early detection of disease, but they often lack the required sensitivity. Here we investigate fluorescent nanodiamonds as an ultrasensitive label for in vitro diagnostics, using a microwave field to modulate emission intensity 5 and frequency-domain analysis 6 to separate the signal from background autofluorescence 7 , which typically limits sensitivity. Focusing on the widely used, low-cost lateral flow format as an exemplar, we achieve a detection limit of 8.2 × 10 -19 molar for a biotin-avidin model, 10 5  times more sensitive than that obtained using gold nanoparticles. Single-copy detection of HIV-1 RNA can be achieved with the addition of a 10-minute isothermal amplification step, and is further demonstrated using a clinical plasma sample with an extraction step. This ultrasensitive quantum diagnostics platform is applicable to numerous diagnostic test formats and diseases, and has the potential to transform early diagnosis of disease for the benefit of patients and populations.

Despite widened access to HIV testing, around half of those infected worldwide are unaware of their HIV-positive status and linkage to care remains a major challenge. Current rapid HIV tests are typically analogue risking incorrect interpretation, no facile electronic data capture, poor linkage to care and data loss for public health. Smartphone-connected diagnostic devices have potential to dramatically improve access to testing and patient retention with electronic data capture and wireless connectivity. We report a pilot clinical study of surface acoustic wave biosensors based on low-cost components found in smartphones to diagnose HIV in 133 patient samples. We engineered a small, portable, laboratory prototype and dual-channel biochips, with in-situ reference control coating and miniaturised configuration, requiring only 6 µL plasma. The dual-channel biochips were functionalized by ink-jet printing with capture coatings to detect either anti-p24 or anti-gp41 antibodies, and a reference control. Biochips were tested with 31 plasma samples from patients with HIV, and 102 healthy volunteers. SH-SAW biosensors showed excellent sensitivity, specificity, low sample volumes and rapid time to result, and were benchmarked to commercial rapid HIV tests. Testing for individual biomarkers found sensitivities of 100% (anti-gp41) and 64.5% (anti-p24) (combined sensitivity of 100%) and 100% specificity, within 5 min. All positive results were recorded within 60 s of sample addition with an electronic readout. Next steps will focus on a smartphone-connected device prototype and user-friendly app interface for larger scale evaluation and field studies, towards our ultimate goal of a new generation of affordable, connected point-of-care HIV tests., Competing Interests: Competing interestsD.A., V.L., R.P., Z.C. and H.Y. are employed by OJ-Bio which produces the SH-SAW prototype. A studentship to V.T. was part-funded by OJ-Bio. The remaining authors declare no competing interests.

Background: Routine influenza surveillance, based on laboratory confirmation of viral infection, often fails to estimate the true burden of influenza-like illness (ILI) in the community because those with ILI often manage their own symptoms without visiting a health professional. Internet-based surveillance can complement this traditional surveillance by measuring symptoms and health behavior of a population with minimal time delay. Flusurvey, the UK's largest crowd-sourced platform for surveillance of influenza, collects routine data on more than 6000 voluntary participants and offers real-time estimates of ILI circulation. However, one criticism of this method of surveillance is that it is only able to assess ILI, rather than virologically confirmed influenza., Objective: We designed a pilot study to see if it was feasible to ask individuals from the Flusurvey platform to perform a self-swabbing task and to assess whether they were able to collect samples with a suitable viral content to detect an influenza virus in the laboratory., Methods: Virological swabbing kits were sent to pilot study participants, who then monitored their ILI symptoms over the influenza season (2014-2015) through the Flusurvey platform. If they reported ILI, they were asked to undertake self-swabbing and return the swabs to a Public Health England laboratory for multiplex respiratory virus polymerase chain reaction testing., Results: A total of 700 swab kits were distributed at the start of the study; from these, 66 participants met the definition for ILI and were asked to return samples. In all, 51 samples were received in the laboratory, 18 of which tested positive for a viral cause of ILI (35%)., Conclusions: This demonstrated proof of concept that it is possible to apply self-swabbing for virological laboratory testing to an online cohort study. This pilot does not have significant numbers to validate whether Flusurvey surveillance accurately reflects influenza infection in the community, but highlights that the methodology is feasible. Self-swabbing could be expanded to larger online surveillance activities, such as during the initial stages of a pandemic, to understand community transmission or to better assess interseasonal activity., (©Clare Wenham, Eleanor R Gray, Candice E Keane, Matthew Donati, Daniela Paolotti, Richard Pebody, Ellen Fragaszy, Rachel A McKendry, W John Edmunds. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 01.03.2018.)

The development is reported of an ultra-rapid, point-of-care diagnostic device which harnesses surface acoustic wave (SAW) biochips, to detect HIV in a finger prick of blood within 10 seconds (sample-in-result-out). The disposable quartz biochip, based on microelectronic components found in every consumer smartphone, is extremely fast because no complex labelling, amplification or wash steps are needed. A pocket-sized control box reads out the SAW signal and displays results electronically. High analytical sensitivity and specificity are found with model and real patient blood samples. The findings presented here open up the potential of consumer electronics to cut lengthy test waiting times, giving patients on the spot access to potentially life-saving treatment and supporting more timely public health interventions to prevent disease transmission.

Biological thiols are antioxidants essential for the prevention of disease. For example, low levels of the tripeptide glutathione are associated with heart disease, cancer, and dementia. Mn 2+ -doped wide bandgap semiconductor nanocrystals exhibit luminescence and magnetic properties that make them attractive for bimodal imaging. We found that these nanocrystals and silica-encapsulated nanoparticle derivatives exhibit enhanced luminescence in the presence of thiols in both organic solvent and aqueous solution. The key to using these nanocrystals as sensors is control over their surfaces. The addition of a ZnS barrier layer or shell produces more stable nanocrystals that are isolated from their surroundings, and luminescence enhancement is only observed with thinner, intermediate shells. Tunability is demonstrated with dodecanethiol and sensitivities decrease with thin, medium, and thick shells. Turn-on nanoprobe luminescence is also generated by several biological thiols, including glutathione, N -acetylcysteine, cysteine, and dithiothreitol. Nanoparticles prepared with different ZnS shell thicknesses demonstrated varying sensitivity to glutathione, which allows for the tuning of particle sensitivity without optimization. The small photoluminescence response to control amino acids and salts indicates selectivity for thiols. Preliminary magnetic measurements highlight the challenge of optimizing sensors for different imaging modalities. In this work, we assess the prospects of using these nanoparticles as luminescent turn-on thiol sensors and for MRI., Competing Interests: The authors declare no conflict of interest.

HIV infection profoundly affects many parameters of the immune system and ultimately leads to AIDS, yet which factors are most important for determining resistance, pathology, and response to antiretroviral treatment - and how best to monitor them - remain unclear. We develop a quantitative high-throughput sequencing pipeline to characterize the TCR repertoires of HIV-infected individuals before and after antiretroviral therapy, working from small, unfractionated samples of peripheral blood. This reveals the TCR repertoires of HIV(+) individuals to be highly perturbed, with considerably reduced diversity as a small proportion of sequences are highly overrepresented. HIV also causes specific qualitative changes to the repertoire including an altered distribution of V gene usage, depletion of public TCR sequences, and disruption of TCR networks. Short-term antiretroviral therapy has little impact on most of the global damage to repertoire structure, but is accompanied by rapid changes in the abundance of many individual TCR sequences, decreases in abundance of the most common sequences, and decreases in the majority of HIV-associated CDR3 sequences. Thus, high-throughput repertoire sequencing of small blood samples that are easy to take, store, and process can shed light on various aspects of the T-cell immune compartment and stands to offer insights into patient stratification and immune reconstitution.

Unlabelled: 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., (Copyright © 2014 Depledge et al.)

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.

Background: Understanding the mechanism by which viruses enter their target cell is an essential part of understanding their infectious cycle. Previous studies have focussed on the multiplicity of viral envelope proteins that need to bind to their cognate receptor to initiate entry. Avian sarcoma and leukosis virus Envelope protein (ASLV Env) mediates entry via a receptor, Tva, which can be attached to the cell surface either by a phospholipid anchor (Tva800) or a transmembrane domain (Tva950). In these studies, we have now investigated the number of target receptors necessary for entry of ASLV Env-pseudotyped virions., Results: Using titration and modelling experiments we provide evidence that binding of more than one receptor, probably two, is needed for entry of virions via Tva800. However, binding of just one Tva950 receptor is sufficient for successful entry., Conclusions: The different modes of attachment of Tva800 and Tva950 to the cell membrane have important implications for the utilisation of these proteins as receptors for viral binding and/or uptake.

Background: Several studies have implicated a recently discovered gammaretrovirus, XMRV (Xenotropic murine leukaemia virus-related virus), in chronic fatigue syndrome and prostate cancer, though whether as causative agent or opportunistic infection is unclear. It has also been suggested that the virus can be found circulating amongst the general population. The discovery has been controversial, with conflicting results from attempts to reproduce the original studies., Methodology/principal Findings: We extracted peripheral blood DNA from a cohort of 540 HIV-1-positive patients (approximately 20% of whom have never been on anti-retroviral treatment) and determined the presence of XMRV and related viruses using TaqMan PCR. While we were able to amplify as few as 5 copies of positive control DNA, we did not find any positive samples in the patient cohort., Conclusions/significance: In view of these negative findings in this highly susceptible group, we conclude that it is unlikely that XMRV or related viruses are circulating at a significant level, if at all, in HIV-1-positive patients in London or in the general population.

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.

Background: Xenotropic 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., Results: Here 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., Conclusions: We 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.

Encoding the global geometric shape of an enclosed environment is a principal means of orientation in human and non-human animals. Animals spontaneously encode the geometry of an enclosure even when featural information is available. Although features can be used, they typically do not overshadow geometry. However, all previously tested organisms have been reared in human-made environments with salient geometrical cues. Here, we show that wild-caught mountain chickadees (Poecile gambeli) do not spontaneously encode the geometry of an enclosure when salient features are present near the goal. However, chickadees trained without salient features encode geometric information, but this encoding is overshadowed by features.