RNA viruses cause significant human pathology and are responsible for the majority of emerging zoonoses. Mainstream diagnostic assays are challenged by their intrinsic diversity, leading to false ...negatives and incomplete characterisation. New sequencing techniques are expanding our ability to agnostically interrogate nucleic acids within diverse sample types, but in the clinical setting are limited by overwhelming host material and ultra-low target frequency. Through selective host RNA depletion and compensatory protocol adjustments for ultra-low RNA inputs, we are able to detect three major blood-borne RNA viruses - HIV, HCV and HEV. We recovered complete genomes and up to 43% of the genome from samples with viral loads of 10
and 10
IU/ml respectively. Additionally, we demonstrated the utility of this method in detecting and characterising members of diverse RNA virus families within a human plasma background, some present at very low levels. By applying this method to a patient sample series, we have simultaneously determined the full genome of both a novel subtype of HCV genotype 6, and a co-infecting human pegivirus. This method builds upon earlier RNA metagenomic techniques and can play an important role in the surveillance and diagnostics of blood-borne viruses.
The long and expanding list of viral pathogens associated with causing encephalitis confounds current diagnostic procedures, and in up to 50% of cases, the etiology remains undetermined. ...Sequence-agnostic metagenomic next-generation sequencing (mNGS) obviates the need to specify targets in advance and thus has great potential in encephalitis diagnostics. However, the low relative abundance of viral nucleic acids in clinical specimens poses a significant challenge. Our protocol employs two novel techniques to selectively remove human material at two stages, significantly increasing the representation of viral material. Our bioinformatic workflow using open source protein- and nucleotide sequence-matching software balances sensitivity and specificity in diagnosing and characterizing any DNA viruses present. A panel of 12 cerebrospinal fluid (CSFs) from encephalitis cases was retrospectively interrogated by mNGS, with concordant results in seven of nine samples with a definitive DNA virus diagnosis, and a different herpesvirus was identified in the other two. In two samples with an inconclusive diagnosis, DNA viruses were detected and in a virus-negative sample, no viruses were detected. This assay has the potential to detect DNA virus infections in cases of encephalitis of unknown etiology and to improve the current screening tests by identifying new and emerging agents.
Choice of direct acting antiviral (DAA) therapy for Hepatitis C Virus (HCV) in the United Kingdom and similar settings usually requires knowledge of the genotype and, in some cases, antiviral ...resistance (AVR) profile of the infecting virus. To determine these, most laboratories currently use Sanger technology, but next-generation sequencing (NGS) offers potential advantages in throughput and accuracy. However, NGS poses unique technical challenges, which require idiosyncratic development and technical validation approaches. This applies particularly to virology, where sequence diversity is high and the amount of starting genetic material is low, making it difficult to distinguish real data from artifacts. We describe the development and technical validation of a sequence capture-based HCV whole genome sequencing (WGS) assay to determine viral genotype and AVR profile. We use clinical samples of known subtypes and viral loads, and simulated FASTQ datasets to validate the analytical performances of both the wet laboratory and bioinformatic pipeline procedures. We show high concordance of the WGS assay compared to current "gold standard" Sanger assays. Specificity was 92.3 and 96.1% for AVR and genotyping, respectively. Discordances were due to the inability of Sanger assays to assign the correct subtype or accurately call mixed drug-resistant variants. We show high repeatability and reproducibility with >99.8% sequence similarity between sequence runs as well as high precision for variant frequency detection at >98.8% in the 95th percentile. Post-sequencing bioinformatics quality control workflows allow the accurate distinction between mixed infections, cross-contaminants and recombinant viruses at a threshold of >5% for the minority population. The sequence capture-based HCV WGS assay is more accurate than legacy AVR and genotyping assays. The assay has now been implemented in the clinical pathway of England's National Health Service HCV treatment programs, representing the first validated HCV WGS pipeline in clinical service. The data generated will additionally provide granular national-level genomic information for public health policy making and support the WHO HCV elimination strategy.
Using deep sequencing technologies such as Illumina's platform, it is possible to obtain reads from the viral RNA population revealing the viral genome diversity within a single host. A range of ...software tools and pipelines can transform raw deep sequencing reads into Sequence Alignment Mapping (SAM) files. We propose that interpretation tools should process these SAM files, directly translating individual reads to amino acids in order to extract statistics of interest such as the proportion of different amino acid residues at specific sites. This preserves per-read linkage between nucleotide variants at different positions within a codon location. The samReporter is a subsystem of the GLUE software toolkit which follows this direct read translation approach in its processing of SAM files. We test samReporter on a deep sequencing dataset obtained from a cohort of 241 UK HCV patients for whom prior treatment with direct-acting antivirals has failed; deep sequencing and resistance testing have been suggested to be of clinical use in this context. We compared the polymorphism interpretation results of the samReporter against an approach that does not preserve per-read linkage. We found that the samReporter was able to properly interpret the sequence data at resistance-associated locations in nine patients where the alternative approach was equivocal. In three cases, the samReporter confirmed that resistance or an atypical substitution was present at NS5A position 30. In three further cases, it confirmed that the sofosbuvir-resistant NS5B substitution S282T was absent. This suggests the direct read translation approach implemented is of value for interpreting viral deep sequencing data.
The NA60 experiment has studied low-mass muon pair production in proton–nucleus collisions with a system of Be, Cu, In, W, Pb and U targets, using a 400 GeV proton beam at the CERN SPS. The ...transverse momentum spectra of the
ρ
/
ω
and
ϕ
mesons are measured in the full
p
T
range accessible, from
p
T
=
0
up to
2
GeV/c
. The nuclear dependence of the production cross sections of the
η
,
ω
and
ϕ
mesons has been found to be consistent with the power law
σ
pA
∝
A
α
, with the
α
parameter increasing as a function of
p
T
for all the particles, and an approximate hierarchy
α
η
≈
α
ϕ
>
α
ω
. The cross section ratios
σ
η
/
σ
ω
,
σ
ρ
/
σ
ω
and
σ
ϕ
/
σ
ω
have been studied as a function of the size A of the production target, and an increase of the
η
and
ϕ
yields relative to the
ω
is observed from p–Be to p–U collisions.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We report on a precision measurement of low-mass muon pairs in 158 AGeV indium-indium collisions at the CERN SPS. A significant excess of pairs is observed above the yield expected from neutral meson ...decays. The unprecedented sample size of 360,000 dimuons and the good mass resolution of about 2% allow us to isolate the excess by subtraction of the decay sources. The shape of the resulting mass spectrum is consistent with a dominant contribution from pi+pi- -->rho -->mu+mu- annihilation. The associated space-time averaged spectral function shows a strong broadening, but essentially no shift in mass. This may rule out theoretical models linking hadron masses directly to the chiral condensate.
The NA60 experiment at the CERN SPS has studied low-mass dimuon production in 158A GeV In-In collisions. An excess of pairs above the known meson decays has been reported before. We now present ...precision results on the associated transverse momentum spectra. The slope parameter Teff extracted from the spectra rises with dimuon mass up to the rho, followed by a sudden decline above. While the initial rise is consistent with the expectations for radial flow of a hadronic decay source, the decline signals a transition to an emission source with much smaller flow. This may well represent the first direct evidence for thermal radiation of partonic origin in nuclear collisions.
NA60 results on thermal dimuons Arnaldi, R.; Banicz, K.; Borer, K. ...
European physical journal. C, Particles and fields,
06/2009, Letnik:
61, Številka:
4
Journal Article, Conference Proceeding
Recenzirano
Odprti dostop
The NA60 experiment at the CERN SPS has measured muon pairs with unprecedented precision in 158
A
GeV In–In collisions. A strong excess of pairs above the known sources is observed in the whole ...mass region 0.2<
M
<2.6 GeV. The mass spectrum for
M
<1 GeV is consistent with a dominant contribution from
π
+
π
−
→
ρ
→
μ
+
μ
−
annihilation. The associated
ρ
spectral function shows a strong broadening, but essentially no shift in mass. For
M
>1 GeV, the excess is found to be prompt, not due to enhanced charm production, with pronounced differences to Drell–Yan pairs. The slope parameter
T
eff
associated with the transverse momentum spectra rises with mass up to the
ρ
, followed by a sudden decline above. The rise for
M
<1 GeV is consistent with radial flow of a hadronic emission source. The seeming absence of significant flow for
M
>1 GeV and its relation to parton–hadron duality is discussed in detail, suggesting a dominantly partonic emission source in this region. A comparison of the data to the present status of theoretical modeling is also contained. The accumulated empirical evidence, including also a Planck-like shape of the mass spectra at low
p
T
and the lack of polarization, is consistent with a global interpretation of the excess dimuons as thermal radiation. We conclude with first results on
ω
in-medium effects.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Hepatitis C virus (HCV) has a high genetic diversity and is classified into 8 genotypes and over 90 subtypes with some endemic to specific world regions. This could compromise direct-acting antiviral ...(DAA) efficacy and global HCV elimination.
We characterised HCV subtypes 'rare' to the UK (non-1a/1b/2b/3a/4d) by whole genome sequencing via a national surveillance programme. Genetic analyses to determine the genotype of samples with unresolved genotypes were undertaken by comparison with ICTV HCV reference sequences.
Two HCV variants were characterised as being closely related to the recently identified genotype 8 (GT8), with >85% pairwise genetic distance similarity to GT8 sequences and within the typical inter-subtype genetic distance range. The individuals infected by the variants were UK residents originally from Pakistan and India. In contrast, a third variant was only confidently identified to be more similar to GT6 compared to other genotypes across 6% of the genome and was isolated from a UK resident originally from Guyana. All three were cured with pangenotypic DAAs (Sofosbuvir + Velpatasvir or Glecaprevir + Pibrentasvir) despite the presence of resistance polymorphisms in NS3 (80 K/168E), NS5A (28 V/30S/62L/92S/93S) and NS5B (159F).
This study expands our knowledge of HCV diversity by identifying two new GT8 subtypes and potentially a new genotype.
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