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Routine childhood vaccination against measles, mumps and rubella has virtually abolished virus-related morbidity and mortality. Notwithstanding this, we describe here devastating neurological complications associated with the detection of live-attenuated mumps virus Jeryl Lynn (MuVJL5) in the brain of a child who had undergone successful allogeneic transplantation for severe combined immunodeficiency (SCID). This is the first confirmed report of MuVJL5 associated with chronic encephalitis and highlights the need to exclude immunodeficient individuals from immunisation with live-attenuated vaccines. The diagnosis was only possible by deep sequencing of the brain biopsy. Sequence comparison of the vaccine batch to the MuVJL5 isolated from brain identified biased hypermutation, particularly in the matrix gene, similar to those found in measles from cases of SSPE. The findings provide unique insights into the pathogenesis of paramyxovirus brain infections. Electronic supplementary material The online version of this article (doi:10.1007/s00401-016-1629-y) contains supplementary material, which is available to authorized users.
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Acta Neuropathol (2017) 133:139–147
DOI 10.1007/s00401-016-1629-y
CASE REPORT
Deep sequencing reveals persistence of cell‑associated mumps
vaccine virus in chronic encephalitis
Sofia Morfopoulou1 · Edward T. Mee2 · Sarah M. Connaughton2 · Julianne R. Brown3 · Kimberly Gilmour4 ·
WK ‘Kling’ Chong5 · W. Paul Duprex6 · Deborah Ferguson2 · Mike Hubank7 · Ciaran Hutchinson8 ·
Marios Kaliakatsos9 · Stephen McQuaid10,11 · Simon Paine8,12 · Vincent Plagnol13 · Christopher Ruis1 ·
Alex Virasami8 · Hong Zhan14 · Thomas S. Jacques8,15 · Silke Schepelmann2 · Waseem Qasim16,17 · Judith Breuer1,3
Received: 11 August 2016 / Revised: 4 October 2016 / Accepted: 4 October 2016 / Published online: 21 October 2016
© The Author(s) 2016. This article is published with open access at Springerlink.com
diagnosis was only possible by deep sequencing of the
brain biopsy. Sequence comparison of the vaccine batch
to the MuVJL5 isolated from brain identified biased hyper-
mutation, particularly in the matrix gene, similar to those
found in measles from cases of SSPE. The findings provide
unique insights into the pathogenesis of paramyxovirus
brain infections.
Introduction
Chronic encephalitis with progressive loss of motor and
cognitive function and high levels of intrathecal antibod-
ies has been associated with persistent measles and rubella
infections of the brain, most commonly in the context of
Abstract Routine childhood vaccination against measles,
mumps and rubella has virtually abolished virus-related
morbidity and mortality. Notwithstanding this, we describe
here devastating neurological complications associated
with the detection of live-attenuated mumps virus Jeryl
Lynn (MuVJL5) in the brain of a child who had undergone
successful allogeneic transplantation for severe combined
immunodeficiency (SCID). This is the first confirmed
report of MuVJL5 associated with chronic encephalitis and
highlights the need to exclude immunodeficient individu-
als from immunisation with live-attenuated vaccines. The
Electronic supplementary material The online version of this
article (doi:10.1007/s00401-016-1629-y) contains supplementary
material, which is available to authorized users.
* Sofia Morfopoulou
sofia.morfopoulou.10@ucl.ac.uk
1 Division of Infection and Immunity, University College
London, Cruciform Building, Gower Street, London WC1E
6BT, UK
2 Virology Division, National Institute for Biological
Standards and Control, Potters Bar EN6 3QG, UK
3 Microbiology, Virology and Infection Control, Great
Ormond Street Hospital for Children, NHS Foundation Trust,
London WC1N 3JH, UK
4 Department of Immunology, Great Ormond Street Hospital,
NHS Foundation Trust, London WC1N 3JH, UK
5 Department of Radiology, Great Ormond Street Hospital
for Children, NHS Foundation Trust, London WC1N 3JH,
UK
6 Department of Microbiology, Boston University School
of Medicine, Boston, MA 02118, USA
7 Genetics and Genomics Medicine UCL Great Ormond Street
Institute of Child Health, London WC1N 1EH, UK
8 Department of Histopathology, Great Ormond Street Hospital
for Children, NHS Foundation Trust, London WC1N 3JH,
UK
9 Neurosciences Department, Great Ormond Street Hospital,
NHS Foundation Trust, London WC1N 3JH, UK
10 Molecular Pathology Programme, Centre for Cancer
Research and Cell Biology, Queen’s University, Belfast, UK
11 Tissue Pathology, Belfast Health and Social Care Trust,
Belfast City Hospital, Lisburn Road, Belfast, UK
12 Nottingham University Hospitals NHS Trust, Queen’s
Medical Centre, Nottingham NG7 2UH, UK
13 UCL Genetics Institute, University College London,
London WC1E 6BT, UK
14 Molecular and Cellular Immunology Section, UCL Great
Ormond Street Institute of Child Health, London WC1N
1EH, UK
15 Developmental Biology and Cancer Programme, UCL Great
Ormond Street Institute of Child Health, London WC1N
1EH, UK
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140 Acta Neuropathol (2017) 133:139–147
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subacute sclerosing pan-encephalitis (SSPE). We describe
here a case of chronic panencephalitis in a child who had
undergone successful allogeneic haematopoietic stem cell
transplantation (allo-SCT) for severe combined immu-
nodeficiency (SCID) in whom neither measles nor other
pathogens could be detected. Using deep sequencing of
fresh brain biopsy material, we identified the Jeryl Lynn
5 mumps virus (MuVJL5), a component of the measles,
mumps, rubella (MMR) vaccine that had been administered
to the child before the diagnosis of SCID. Similar to find-
ings in measles viruses recovered from cases of SSPE, the
mumps virus genome from the brain showed evidence of
biased hypermutation, particularly in the matrix (M) gene.
Comparison with sequence data from the original vaccine
batch used to immunise this child identified the expansion
of variants present at low frequency in the vaccine as well
as de novo fixed missense substitutions. This case repre-
sents the first conclusive demonstration of chronic panen-
cephalitis due to mumps virus.
Case report
An 18-month-old male infant of consanguineous parents
was diagnosed with SCID due to recombinase activat-
ing gene 1 (RAG1) deficiency (Fig. 1a) 4 months after
MMR vaccination. The infant received a CD34-selected
haploidentical allo-SCT. Full donor chimerism was rap-
idly achieved, with recovery of a diverse T cell repertoire
(Fig. 1a) and excellent thymopoiesis. Post-transplant auto-
immune cytopenias necessitated rituximab therapy, fol-
lowing which immunoglobulin replacement therapy was
administered. Six months post-allo-SCT the child devel-
oped a febrile illness with rash, diarrhoea, lethargy and
seizures, with evidence of encephalitis on magnetic reso-
nance imaging (MRI) with raised CSF protein (1.35 g/L)
and 11 lymphocytes. Despite extensive screening for neu-
rotropic viruses and bacteria (Table S1), no pathogen was
detected. He was treated with antimicrobials, antivirals and
steroids, making a good recovery, and was discharged on
anticonvulsant therapy. Over the next few months, the child
was noted to have behavioural problems, hearing impair-
ment and speech and language delay. One year after dis-
charge, the seizures recurred with only partial response to
antiepileptic treatment, but he remained stable for another
9 months. Repeat MRI scan 2 years after initial encepha-
litic illness showed no new lesions. Over the next few
months, the child’s neurological condition deteriorated,
with increasing seizures together with episodes of lethargy,
disorientation, agitation, ataxic gait, visual loss and even-
tual hospitalisation.
Repeat MRI of the brain at 40 months post-allo-SCT
revealed abnormalities of grey and white matter, involving
both cerebral hemispheres with multiple foci of contrast
enhancement, including the basal ganglia, temporal and
parieto-occipital cortices (Fig. 1b). In the absence of a firm
diagnosis, a brain biopsy was taken.
Broad-spectrum antibiotics, aciclovir, ganciclovir and
antifungal therapy were administered, as well as intrave-
nous immunoglobulin (IVIG) and high-dose methyl pred-
nisolone. Increasing seizures, left-sided weakness, cortical
blindness and progressive global neurological deterioration
over several weeks ended with the patient’s death 7 weeks
following his last hospital admission.
Materials and methods
PCR assays
The viruses listed in Table S1 were tested for using real-
time PCR and found to be negative using specific primers
and TaqMan probes on an ABI 7500 thermocycler.
All PCR assays were performed in-house at GOSH
(Great Ormond Street Hospital for Children, London) as
part of the routine diagnostic service, unless indicated oth-
erwise, in which case they were sent to a different labora-
tory for testing.
The CSF and urine samples collected during the patient’s
last hospitalisation, as well as RNA re-extracted from the
brain biopsy, were sent to the Public Health England Virus
Reference Laboratory (Colindale) for mumps and mumps
vaccine-specific RT-PCR (targeting the SH and HN gene)
as well as measles and rubella-specific RT-PCR.
Sequencing
Library preparation—brain biopsy
Total RNA was purified from the frozen brain biopsy and
polyA RNA-separated for sequencing library preparation.
Samples were sequenced on the Illumina NextSeq500
16 Great Ormond Street Hospital for Children, NHS Foundation
Trust, Molecular and Cellular Immunology, London WC1N
3JH, UK
17 UCL Great Ormond Street Institute of Child Health,
London WC1N 1EH, UK
Fig. 1 a Patient timeline presenting important clinical events
(m.o. = months old) and immune recovery of CD3+ CD4+ T cells
over this period. b MRI brain scan showing coronal T2-weighted
FLAIR (FLuid Attenuated Inversion Recovery) and axial post-con-
trast T1-weighted images with bilateral basal ganglia lesions (white
arrows) and enhancing cortical and deep grey matter lesions (black
arrows). The pattern was typical of those described in subacute
panencephalitis
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141Acta Neuropathol (2017) 133:139–147
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a
b
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142 Acta Neuropathol (2017) 133:139–147
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instrument (Illumina, San Diego, US) using an 81 bp
paired-end run. Libraries to be multiplexed in the same run
were pooled in equimolar quantities, calculated from qPCR
and/or Bioanalyser fragment analysis.
Samples were processed using Illumina’s TruSeq
Stranded mRNA LT sample preparation kit (p/n RS-122-
2101) according to the manufacturer’s instructions. Devia-
tions from the protocol were as follows: (1) 250 ng total
RNA was used as starting material. (2) Fragmentation was
carried out for 10 min instead of 8 min. (3) 14 Cycles of
PCR were used.
Briefly, mRNA was isolated from total RNA using
oligo dT beads to pull down poly-adenylated transcripts.
The purified mRNA was fragmented using chemical frag-
mentation (heat and divalent metal cation) and primed
with random hexamers. Strand-specific first-strand cDNA
was generated using SuperScript II Reverse Transcriptase
(Life Technologies) and actinomycin D. This allows RNA-
dependent synthesis while preventing spurious DNA-
dependent synthesis. The second cDNA strand was marked
by performing synthesis incorporating dUTP.
The cDNA is then “A-tailed” at the 3 end to pre-
vent self-ligation during the addition of the full-length
TruSeq Adaptors (adaptors have a complementary “T”
overhang). The adaptors contain sequences that allow the
libraries to be amplified by PCR, bind to the flow cell and
be uniquely identified by way of a 6 bp index sequence.
Finally, a PCR is carried out to amplify only those cDNA
fragments that have adaptors bound to both ends.
Library preparation—vaccine
An archived vial from the batch of MMR vaccine used to
immunise the child was deep sequenced in a 2 × 251 paired-
end sequencing on an Illumina MiSeq as part of a 16-sam-
ple pool, generating 1,570,733 read pairs, at the National
Institute for Biological Standards and Control (NIBSC).
Lyophilised vaccine was resuspended in 500 µl ster-
ile water for injection and viral RNA extracted using the
QIAamp Viral RNA Mini Spin Kit (Qiagen) according to
the method of the manufacturer, with the omission of car-
rier RNA. DNA libraries were prepared by random reverse-
transcriptase polymerase chain reaction as described pre-
viously [18], followed by fragmentation, adaptor addition
and indexing using the Nextera XT library preparation kit
(Illumina).
Immunohistochemistry
Formalin-fixed paraffin-embedded sections of brain biopsy
were examined by conventional histology. Immunohisto-
chemistry was undertaken using two different antibodies in
two independent laboratories.
Immunohistochemistry was undertaken using mumps
nucleoprotein (N) antibody (7B10: sc-57921 Santa Cruz) at
1 in 25 using a Leica Bond Max automated staining sys-
tem with pretreatment HIER (30) ER2. Additional immu-
nohistochemistry was independently undertaken using a
different antibody. The monoclonal mumps antibody used
[16] recognises the N protein of MuV (N93-51/01) and
was used on an automated Leica BondMax immunostainer
at a dilution of 1/4000 following HIER2 pre-treatment for
20 min. Detection sites were detected with a polymer-based
detection system (Bond, Newcastle upon Tyne, UK, Cat.
No. DS9800). Detection of mumps N in the CNS by immu-
nohistochemistry is shown in Figure S2.
Bioinformatics analysis
For the analysis of the brain biopsy sequencing data, we
implemented the following steps. We first removed dupli-
cate sequences that can arise from PCR amplification with
an in-house script that collapses pairs of reads based on
sequence identity using 90 % of the sequence as signature
(20 % removed as duplicates). Half of the reads overlapped
with their “mates” within pairs and we therefore merged
the overlapping reads using PEAR [32], taking into account
both sequence match and quality scores. We performed
quality control using PrinSeq [25], trimming low-quality
ends and removing reads that had average quality less than
15. We subsequently removed human sequences, using a
quick aligner (Novoalign version V2.07.13—human refer-
ence genome GRCh37) as well as BLASTn [2]. We per-
formed de novo assembly of high-quality contigs using
Velvet [31] (kmer = 81). Finally, we annotated the contigs
and the unassembled reads against a custom protein refer-
ence database using BLASTx. Our custom protein refer-
ence database consists of viral, bacterial, human and mouse
RefSeq proteins. More specifically, all known viruses in
the RefSeq collection are used ftp://ftp.ncbi.nlm.nih.gov/
refseq/release/viral/viral.1.protein.faa.gz, as well as all the
bacteria of the human microbiome, according to ftp://ftp.
ncbi.nih.gov/genomes/HUMAN_MICROBIOM/Bacteria/
all.faa.tar.gz. The BLASTx results were the input of meta-
Mix [21].
For the analysis of the vaccine sequencing, we first
removed 15 % of the reads as duplicates and merged over-
lapping reads. We trimmed the reads based on base quality
(q = 20) using Trim Galore! (http://www.bioinformatics.
babraham.ac.uk/projects/trim_galore/). We then selected
for Jeryl Lynn vaccine strain reads using BLASTn [2] and a
Jeryl Lynn nucleotide reference database.
We performed de novo assembly with SPADeS [4] fol-
lowed by QUAST [12] for both the vaccine and the brain
sequencing data. In the latter case, we used the MuV reads
as identified by metaMix [21]. Novoalign, Samtools [17]
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143Acta Neuropathol (2017) 133:139–147
1 3
and VarScan2 [15] were used for consensus sequence gen-
eration and variant calling. We filtered variants based on
quality, depth, frequency and strand bias (quality 30, at
least 5 reads for the variant site, frequency 5 %, p value
<0.01). The variants were annotated with SnpEff [7].
We compared the number of non-synonymous changes
observed in each of the MuV genes to the number we
would expect if the observed missense mutations were ran-
domly distributed across the genome, correcting for the
gene length. Significant deviation from the expected num-
ber of mutations was tested with the goodness-of-fit two-
tailed exact binomial test. Analysis was conducted with the
statistical language R (http://www.r-project.org).
We estimated a maximum likelihood phylogenetic tree
using RAxML [27] and 64 full MuV genomes from Gen-
Bank (accessed on 20 June 2015).
Results
CSF on last admission was acellular, but with oligoclonal
bands, although total protein was low 0.12 g/L (normal
0.15–0.45 g/L. The oligoclonal bands were negative for
HSV and VZV antibodies. Polyoma JC virus was detected
by PCR in blood and urine on one occasion each (Table
S2). CSF PCR was negative for 16S (bacterial) and 18S
(fungal) rRNA and for a wide number of viral pathogens
(Table S2), including JC virus, mumps, measles and rubella
viruses. No pathogens were detected in stool, urine or
blood (Table S2). CSF was negative for rubella and mea-
sles antibodies. In the absence of a firm diagnosis, a brain
biopsy showed neuronal loss, astrocytic gliosis, reactive
astrocytes, microglia and chronic inflammatory cells, but
no viral inclusions or granulomata (Fig. 2a–e legend for
detailed description). Infiltrating T lymphocytes were iden-
tified by CD3 staining (Fig. 2f).
RNA-Seq of the brain biopsy [5, 20] resulted in 110 mil-
lion 81 base pair (bp) paired-end reads. Approximately,
one million reads were identified as non-human and were
subsequently used for potential pathogen identification
by the metaMix method [21]. Mumps virus was the only
potential pathogen detected, with 77,624 assigned reads.
The remaining reads were either unclassified or mapped to
human and bacteria that are either environmental, part of
human flora, kit contaminants [23] or of unknown signifi-
cance (Table S3).
No reads mapped to measles, rubella or polyoma JC
viruses. Using de novo assembly, the full-length viral
sequence was recovered (99.94 % genome coverage,
median coverage depth: 290) (Figure S1). Maximum like-
lihood phylogenetic analysis showed that the consensus
viral sequence clustered closely with the MuVJL5 vaccine
strains (Figure S2) and shared 99.6 % identity with the
publicly available sequence (GenBank: FJ211585) of the
mumps component of the MMR preparation administered
to the child. The sequence identified in this study is named
MuVJL5-London (GenBank: KX223397). PCR of RNA
extracted from the brain biopsy confirmed the presence of
MuVJL5 vaccine strain (Table S2), but was negative for all
other viruses, including polyoma JC (Table S2). Immuno-
histochemistry showed the presence of MuV nucleocapsid
(N) protein with a neuronal pattern of staining (Fig. 2g,
h). Control cortex and white matter were negative. We
observed no specific staining for other pathogens (Fig. 2g,h
for details) and no evidence of viral intracytoplasmic or
intranuclear inclusion bodies. Retrospective testing showed
weakly positive mumps antibody in the CSF.
To investigate this finding further, three million 251 bp
paired-end reads were generated from the MMR vaccine
batch that was used to immunise the child, of which 82,000
reads mapped to the MuVJL5 vaccine strain (median cov-
erage: 706, Figure S1). There were no fixed differences
identified compared with the reference FJ211585. Fifteen
positions in the vaccine sequence were found to be poly-
morphic (variant allele frequency (VAF) 5 % or greater,
Table S4).
Of 55 fixed nucleotide differences between MuVJL5-
London and the vaccine, 12 had been present as a minor-
ity variant (10–24 %) in the vaccine (Figure S3, Table S4,
VAF 75 % or greater). Of the remaining 43 new mutations,
28 coded for missense amino acid substitutions (Fig. 3,
Table S5), with nine (32 %) located in the M protein, more
than expected by chance (two-tailed exact binomial test
p = 2.8e04, Table S6). Other than position 140 in the M
protein, the missense mutations were unique to MuVJL5-
London (Figure S6). The Tyr140His substitution in
MuVJL5-London is found in all wild-type strains as well as
the more neurovirulent MuVUr Urabe and the MuVJL2 Jeryl
Lynn 2 vaccine strain, which is a minor component of some
vaccines [1] (Figure S6).
Discussion
Prior to the introduction of routine MMR vaccination,
mumps virus was the commonest cause of meningoen-
cephalitis in children and was associated with serious
sequelae including deafness and orchitis [11, 22]. No
meningoencephalitis has been confirmed following the
Jeryl lynn mumps vaccine, which this child received, and
other adverse effects are rare [19] and usually self-limiting
[14]. Mumps and rubella vaccine virus transcripts were
detected in the brain of a child with IFNAR2 deficiency
who developed fatal encephalitis following MMR vaccina-
tion [9], but brain immunohistochemistry was negative in
this case (JB personal communication).
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144 Acta Neuropathol (2017) 133:139–147
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The presentation of this case, shares features with two
previously published cases of MuV-related progressive
encephalitis [13, 28]. Moreover, like measles SSPE and
measles inclusion body encephalitis (MIBE) [29], which
occurs in immunocompromised children within months of
measles infection or vaccination, and unlike acute MuV
encephalitis, no virus was detected in the CSF. How-
ever, other hallmarks of SSPE and MIBE such as high
virus-specific antibody levels in CSF [29], were absent,
although the child had deficient B cell function.
Importantly, however, in comparison with MuVJL5 from
the vaccine batch used to immunise the child, MuVJL5-
London showed evidence of T to C biased hypermutation,
particularly in the M (matrix) gene suggestive of adenosine
deaminase (ADAR)-driven mutation [10] (Table S4). The
same finding in MV recovered from SSPE and MIBE [6,
Fig. 2 Much of the cortex
showed significant tissue dam-
age with neuronal loss. There
was prominent reactive gliosis
composed of plump astrocyto-
sis with abundant eosinophilic
cytoplasm and immunoreactiv-
ity for glial fibrillary acidic
protein (GFAP) (a, e) with
relative sparing of the superfi-
cial layers of the cortex. Some
areas showed perivascular
lymphocytosis and collections
of macrophages (a, c). In others,
with better neuronal preserva-
tion, there were foci of micro-
glial nodules, neuronophagia,
generalised microgliosis (dem-
onstrated on CD68 staining b,
d)) and patchy parenchymal and
perivascular lymphocytes, the
majority of which were positive
for the T cell markers (f). Occa-
sional foci of mineralisation
were observed, but there was
no vasculitis and no viral inclu-
sions. The pathological changes
extended into the underlying
white matter (data not shown),
which showed patchy loss of
myelin staining on luxol fast
blue. There was focal chronic
inflammation in the leptomenin-
ges. Mumps immunohistochem-
istry was positive in a neuronal
pattern (g, h). We observed
no specific staining for other
pathogens HSV1 or 2, CMV,
EBV, toxoplasma, JC virus, bac-
teria (Gram and Gram Twort),
acid-fast bacteria (Ziehl-
Neelson) or fungi (Grocott),
and no evidence of intracellular
or intranuclear viral inclusion
bodies. a, b Haematoxylin
and eosin (H&E), c, d CD68
immunohistochemistry, e GFAP
immunohistochemistry, f CD3
immunohistochemistry. Scale
bars-b, h 50 μm, a, c, d, e, g
100 μm, f 200 μm
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145Acta Neuropathol (2017) 133:139–147
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29, 30] has been shown to reduce M protein expression [29]
and is mooted to promote cell to cell spread and reduced
viral assembly and shedding into the CSF. Biased hyper-
mutation and lack of protein expression may also reflect the
dispensability of M protein for neurovirulence [3].
MuVJL5-London was characterised by the expansion of
variants present at low frequency in the original vaccine,
two of which have previously been described [8], as well
as fixed de novo mutations in the M, N, P, L and F genes,
a finding which again references those described for MV
in SSPE. While most amino acid substitutions occurred in
the M gene, in vitro data using the rat model implicates the
F protein [3, 16] as key for mumps virus neurovirulence in
acute encephalitis [16, 24] as well as for measles neurovir-
ulence in SSPE [3]. The absence of within-host sequence
diversity in the M and F ORFs (Figure S5, Table S7) fur-
ther suggests functional constraints on these genes and
supports directional selection acting on one or more of the
amino acid changes in these proteins favouring spread and
replication of MuVJL5 in the brain.
In this child, clinical deterioration occurred following
good T cell reconstitution in the context of normal T cell
receptor repertoire and good levels of thymopoiesis. One
possibility is that the presence of mumps tolerised and dys-
regulated T cell responses, thus permitting long-term viral
persistence and the accumulation of pathogenic mutations.
We made use of data on the donor’s HLA genotype to pre-
dict specific CTL cell epitopes (IEDB epitope prediction
tool, percentile rank cutoff 1 %) in the MuVJL5 mumps
vaccine virus. Five missense mutations were located in the
predicted CTL epitopes for the donor’s HLA (Fig. 3, Table
S8). Of these, four had predicted T cell binding affinities
at a level likely to correlate with T cell recognition [26]
and reduced binding affinity in the mutated peptide (Table
S8). This raises the possibility that partial escape of virus
from immune surveillance through specific mutational
changes may have occurred, especially as the same was
not observed for peptides recognised by randomly selected
HLA alleles (Supplementary Appendix, Table S8).
In summary, we report a case of progressive chronic
encephalitis in a patient with SCID, in which the MuVJL5
vaccine strain was detected in brain biopsy by deep
sequencing. This case emphasises the generally poor rates
of pathogen detection in encephalitides, making a strong
case for deep sequencing of brain tissue where other meth-
ods have failed. The similar pattern of viral mutations in
this case and those of measles SSPE suggest a common
pathogenic process and further work is currently under-
way to determine the contribution of fixed mutations to the
chronic encephalitic phenotype observed in the patient. It is
important to note that MMR continues to be a highly effec-
tive and safe vaccine in the vast majority of individuals.
However, this case highlights the importance of developing
strategies such as newborn screening to exclude the very
small proportion of infants at extremely high risk of com-
plications from live-attenuated vaccines.
Fig. 3 Fixed (defined to have frequency greater than 75 %) missense
substitutions in the MuVJL5-London brain mumps virus genome. The
amino acid changes are plotted along the genome, colour-coded for
each viral protein. The black dashed lines are the missense changes
pre-existing as minor variants in the genome of the vaccine virus. The
predicted CTL epitopes are also indicated
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146 Acta Neuropathol (2017) 133:139–147
1 3
Acknowledgments The authors acknowledge Public Health Eng-
land (PHE) and Micropathology Laboratory Ltd. for PCR assays, as
detailed in Supplementary Table 2 and Tony Brooks at UCL Genom-
ics for sequencing services.
Financial disclosure This work was supported by the National
Institute for Health Research (NIHR) Biomedical Research Centre
(BRC) at Great Ormond Street Hospital for Children NHS Founda-
tion Trust (GOSH) and University College London. S.M. was funded
by Pathseek FP7. J. Brown is supported by an NIHR fellowship
and the GOSH/UCL BRC. WPD received grant funding from NIH
R01AI099100. TJ received funding from GOSH Children’s Charity,
The Brain Tumour Charity, Children with Cancer and the GOSH/
ICH Biomedical Research Centre. W.Q received grant funding from
NIHR, GOSH Charity, GOSH/UCL BRC. JB received funding from
the NIHR BRC at University College London Hospitals (UCLH)/
UCL. The remaining authors have nothing to disclose.
Open Access This article is distributed under the terms of the Crea-
tive Commons Attribution 4.0 International License (http://crea-
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
References
1. Afzal MA, Pickford AR, Forsey T, Heath AB, Minor PD
(1993) The Jeryl Lynn vaccine strain of mumps virus is a mix-
ture of two distinct isolates. J Gen Virol 74(Pt 5):917–920.
doi:10.1099/0022-1317-74-5-917
2. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990)
Basic local alignment search tool. J Mol Biol 215:403–410.
doi:10.1016/S0022-2836(05)80360-2
3. Ayata M, Tanaka M, Kameoka K, Kuwamura M, Takeuchi K,
Takeda M, Kanou K, Ogura H (2016) Amino acid substitutions
in the heptad repeat A and C regions of the F protein responsible
for neurovirulence of measles virus Osaka-1 strain from a patient
with subacute sclerosing panencephalitis. Virology 487:141–149.
doi:10.1016/j.virol.2015.10.004
4. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M,
Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD,
Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA,
Pevzner PA (2012) SPAdes: a new genome assembly algorithm
and its applications to single-cell sequencing. J Comput Biol
19:455–477. doi:10.1089/cmb.2012.0021
5. Brown JR, Morfopoulou S, Hubb J, Emmett WA, Ip W, Shah
D, Brooks T, Paine SML, Anderson G, Virasami A, Tong CYW,
Clark DA, Plagnol V, Jacques TS, Qasim W, Hubank M, Breuer J
(2015) Astrovirus VA1/HMO-C: an increasingly recognized neu-
rotropic pathogen in immunocompromised patients. Clin Infect
Dis 60(6):881–888. doi:10.1093/cid/ciu940
6. Cattaneo R, Schmid A, Eschle D, Baczko K, ter Meulen V,
Billeter MA (1988) Biased hypermutation and other genetic
changes in defective measles viruses in human brain infections.
Cell 55:255–265. doi:10.1016/0092-8674(88)90048-7
7. Cingolani P, Platts A, Wang LL, Coon M, Nguyen T, Wang L,
Land SJ, Ruden DM, Lu X (2012) A program for annotating
and predicting the effects of single nucleotide polymorphisms,
SnpEff. Fly (Austin) 6(2):1–13. doi:10.4161/fly.19695
8. Connaughton SM, Wheeler JX, Vitková E, Minor P, Vit-
kova E, Minor P, Schepelmann S (2015) In vitro and in vivo
growth alter the population dynamic and properties of a Jeryl
Lynn mumps vaccine. Vaccine 33:4586–4593. doi:10.1016/j.
vaccine.2015.06.109
9. Duncan CJA, Mohamad SMB, Young DF, Skelton AJ, Leahy TR,
Munday DC, Butler KM, Morfopoulou S, Brown JR, Hubank
M, Connell J, Gavin PJ, McMahon C, Dempsey E, Lynch NE,
Jacques TS, Valappil M, Cant AJ, Breuer J, Engelhardt KR,
Randall RE, Hambleton S (2015) Human IFNAR2 deficiency:
Lessons for antiviral immunity. Sci Transl Med 7:307ra154-
307ra154. doi:10.1126/scitranslmed.aac4227
10. Fehrholz M, Kendl S, Prifert C, Weissbrich B, Lemon K, Ren-
nick L, Duprex PW, Rima BK, Koning FA, Holmes RK, Malim
MH, Schneider-Schaulies J (2012) The innate antiviral factor
APOBEC3G targets replication of measles, mumps and respira-
tory syncytial viruses. J Gen Virol 93:565–576. doi:10.1099/
vir.0.038919-0
11. Galazka AM, Robertson SE, Kraigher A (1999) Mumps and
mumps vaccine: a global review. Bull World Health Organ 77:3–
14. doi:10.1007/BF02727158
12. Gurevich A, Saveliev V, Vyahhi N, Tesler G (2013) QUAST:
quality assessment tool for genome assemblies. Bioinformatics
29:1072–1075. doi:10.1093/bioinformatics/btt086
13. Haginoya K, Ike K, Iinuma K, Yagi T, Kon K, Yokoyama HNY
(1995) Chronic progressive mumps virus encephalitis in a child.
Lancet 346:50
14. Hviid A, Rubin S, Mühlemann K (2008) Seminar: mumps. Lan-
cet 371:932–944. doi:10.1016/S0140-6736(08)60419-5
15. Koboldt DC, Zhang Q, Larson DE, Shen D, Mclellan MD, Lin
L, Miller CA, Mardis ER, Ding L, Wilson RK (2012) VarScan
2 : Somatic mutation and copy number alteration discovery in
cancer by exome sequencing. Genome Res 22(3):568–576.
doi:10.1101/gr.129684.111
16. Lemon K, Rima BK, McQuaid S, Allen IV, Duprex WP
(2007) The F gene of rodent brain-adapted mumps virus is a
major determinant of neurovirulence. J Virol 81:8293–8302.
doi:10.1128/JVI.00266-07
17. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N,
Marth G, Abecasis G, Durbin R (2009) The sequence align-
ment/map format and SAMtools. Bioinformatics 25:2078–2079.
doi:10.1093/bioinformatics/btp352
18. Mee ET, Minor PD, Martin J (2015) High resolution identity
testing of inactivated poliovirus vaccines. Vaccine 33:3533–
3541. doi:10.1016/j.vaccine.2015.05.052
19. Miller E, Andrews N, Stowe J, Grant A, Waight P, Taylor B
(2007) Risks of convulsion and aseptic meningitis following
measles-mumps-rubella vaccination in the United Kingdom. Am
J Epidemiol 165:704–709. doi:10.1093/aje/kwk045
20. Morfopoulou S, Brown JR, Davies EG, Anderson G, Virasami
A, Qasim W, Chong WK, Hubank M, Plagnol V, Desforges
M, Jacques TS, Talbot PJ, Breuer J (2016) Human coronavirus
OC43 associated with fatal encephalitis. N Engl J Med 375:497–
498. doi:10.1056/NEJMc1509458
21. Morfopoulou S, Plagnol V (2015) Bayesian mixture analysis for
metagenomic community profiling. Bioinformatics 31:2930–
2938. doi:10.1093/bioinformatics/btv317
22. Peltola H, Kulkarni PS, Kapre SV, Paunio M, Jadhav SS, Dhere
RM (2007) Mumps outbreaks in Canada and the United States:
time for new thinking on mumps vaccines. Clin Infect Dis
45:459–466. doi:10.1086/520028
23. Salter SJ, Cox MJ, Turek EM, Calus ST, Cookson WO, Moffatt
MF, Turner P, Parkhill J, Loman NJ, Walker AW (2014) Reagent
and laboratory contamination can critically impact sequence-
based microbiome analyses. BMC Biol 12:87. doi:10.1186/
s12915-014-0087-z
24. Sauder CJ, Zhang CX, Ngo L, Werner K, Lemon K, Duprex
WP, Malik T, Carbone K, Rubin SA (2011) Gene-specific
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
147Acta Neuropathol (2017) 133:139–147
1 3
contributions to mumps virus neurovirulence and neuroattenua-
tion. J Virol 85:7059–7069. doi:10.1128/JVI.00245-11
25. Schmieder R, Edwards R (2011) Quality control and preproc-
essing of metagenomic datasets. Bioinformatics 27:863–864.
doi:10.1093/bioinformatics/btr026
26. Sette A, Vitiello A, Reherman B, Fowler P, Nayersina R, Kast
WM, Melief CJ, Oseroff C, Yuan L, Ruppert J, Sidney J, del
Guercio MF, Southwood S, Kubo RT, Chesnut RW, Grey HM,
Chisari FV (1994) The relationship between class I binding affin-
ity and immunogenicity of potential cytotoxic T cell epitopes. J
Immunol 153:5586–5592
27. Stamatakis A (2014) RAxML version 8: a tool for phylogenetic
analysis and post-analysis of large phylogenies. Bioinformatics
30:1312–1313. doi:10.1093/bioinformatics/btu033
28. Vaheri A, Julkunen IKM (1982) Chronic encephalomyelitis
with specific increase in intrathecal mumps antibodies. Lancet
2:685–688
29. Wilson MR, Ludlow M, Duprex WP (2013) Human paramyxovi-
ruses and infections of the central nervous system. In: Singh SK,
Ruzek D (eds) Neuroviral infect. CRC Press, USA, pp 341–372
30. Wong TC, Ayata M, Hirano A, Yoshikawa Y, Tsuruoka H, Yaman-
ouchi K (1989) Generalized and localized biased hypermutation
affecting the matrix gene of a measles virus strain that causes
subacute sclerosing panencephalitis. J Virol 63:5464–5468
31. Zerbino DR, Birney E (2008) Velvet: algorithms for de novo
short read assembly using de Bruijn graphs. Genome Res
18:821–829. doi:10.1101/gr.074492.107
32. Zhang J, Kobert K, Flouri T, Stamatakis A (2014) PEAR: a fast
and accurate Illumina Paired-End reAd mergeR. Bioinformatics
30:614–620. doi:10.1093/bioinformatics/btt593
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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2.
3.
4.
5.
6.
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... Among these, the genotype A strain (Genbank no. KX223397) was special in that it was obtained from a brain tissue specimen of a child who died of chronic encephalitis in 2014 and the strain belonged to the Jeryl Lynn 5 (JL5)-like strain of the MMR vaccine (33). The genotype G strain was widespread and has been reported in the Americas (USA and Canada), Europe (Netherlands), Australia (Australia and New Zealand), Asia (India and Japan), and Africa (Gabon) between 2010 and 2019. ...
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Mumps, a disease caused by the mumps virus (MuV), has been spread widely across the world, especially among children and adolescents. Recent frequent local mumps outbreaks were reported worldwide, which may be caused by the decline in the neutralization ability of the existing attenuated live mumps vaccines against circulating MuV strains which were different from the genotype A or B vaccine strains. There is an urgent need to understand the genotypes of MuV strains currently circulated globally and in China. The gene sequences of MuV strains circulated globally were collected and phylogenetic trees were constructed using different strategies. The results showed that the MuV strains previously circulated globally were predominantly genotype G, while genotype F was predominantly circulated in China, followed by genotype G. The molecular evolution of genotype F MuV strains circulated in China is at a low genetic mutation rate, and the analysis of population dynamics pattern indicates that the incidence of genotype F mumps in China showed a rebound trend. These findings provide a basis for the selection or design of vaccine strains, and the decision of the evaluation strains for immunogenicity and protective efficacy, which laid the foundation for the research and development, as well as the application of next-generation MuV vaccines.
... For example, for viruses that are assembled and released from the cell surface, mutations that limit or prevent cell surface expression of viral proteins can prevent recognition by antibodies. In the measles virus-induced late disease SSPE, virion proteins required for particle assembly at the plasma membrane (hemagglutinin, fusion, and matrix) have acquired changes that prevent cell surface expression and virion assembly but promote cell-to-cell ribonucleoprotein transfer to uninfected cells, thereby allowing continued spread of viral RNA without producing infectious virions [121][122][123][124]. Similar mutations have been observed in the viral RNAs from persistent CNS infections due to mumps and mouse hepatitis viruses [113,125]. ...
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DNA viruses often persist in the body of their host, becoming latent and recurring many months or years later. By contrast, most RNA viruses cause acute infections that are cleared from the host as they lack the mechanisms to persist. However, it is becoming clear that viral RNA can persist after clinical recovery and elimination of detectable infectious virus. This persistence can either be asymptomatic or associated with late progressive disease or nonspecific lingering symptoms, such as may be the case following infection with Ebola or Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Why does viral RNA sometimes persist after recovery from an acute infection? Where does the RNA come from? And what are the consequences?
... For samples processed at the Great Ormond Street Hospital, London (GOSH), mRNA from the three brain biopsy samples was sequenced on an Illumina NextSeq500 instrument using an 81 bp paired-run after library preparation using Illumina's TruSeq Stranded mRNA LT sample preparation kit (p/n RS-122-2101) according to the manufacturer's instructions [19]. The other samples were spiked with Equine Arteritis Virus (EAV) and Phocid Herpes Virus (PhHV) internal controls preceding total nucleic acid extraction using the MagNAPure 96 DNA and Viral NA Small Volume Kit (Roche Diagnostics, Almere, the Netherlands) and sequenced on Illumina NextSeq500 (respiratory samples) or Nova-Seq6000 (CSF samples, plasma) instruments using 150 bp paired-end runs after library preparation using New England BioLabs' NEBNext Ultra Directional RNA Library preparation kit for Illumina with in-house adaptations in order to enable simultaneous detection of both DNA and RNA viruses, at the Leiden University Medical Center (LUMC) [4,20]. ...
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Introduction: Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories. Methods: Metagenomic datasets from 13 clinical samples from patients with encephalitis or viral respiratory infections characterized by PCR were selected. The datasets were analyzed with 13 different pipelines currently used in virological diagnostic laboratories of participating ENNGS members. The pipelines and classification tools were: Centrifuge, DAMIAN, DIAMOND, DNASTAR, FEVIR, Genome Detective, Jovian, MetaMIC, MetaMix, One Codex, RIEMS, VirMet, and Taxonomer. Performance, characteristics, clinical use, and user-friendliness of these pipelines were analyzed. Results: Overall, viral pathogens with high loads were detected by all the evaluated metagenomic pipelines. In contrast, lower abundance pathogens and mixed infections were only detected by 3/13 pipelines, namely DNASTAR, FEVIR, and MetaMix. Overall sensitivity ranged from 80% (10/13) to 100% (13/13 datasets). Overall positive predictive value ranged from 71-100%. The majority of the pipelines classified sequences based on nucleotide similarity (8/13), only a minority used amino acid similarity, and 6 of the 13 pipelines assembled sequences de novo. No clear differences in performance were detected that correlated with these classification approaches. Read counts of target viruses varied between the pipelines over a range of 2-3 log, indicating differences in limit of detection. Conclusion: A wide variety of viral metagenomic pipelines is currently used in the participating clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implicating the need for standardization and validation of metagenomic analysis for clinical diagnostic use. Future studies should address the selective effects due to the choice of different reference viral databases.
... For samples processed at the Great Ormond Street Hospital, London (GOSH), mRNA from the three brain biopsy samples was sequenced on an Illumina NextSeq500 instrument using an 81 bp paired-run after library preparation using Illumina's TruSeq Stranded mRNA LT sample preparation kit (p/n RS-122-2101) according to the manufacturer's instructions [19]. The other samples were spiked with Equine Arteritis Virus (EAV) and Phocid Herpes Virus (PhHV) internal controls preceding total nucleic acid extraction using the MagNAPure 96 DNA and Viral NA Small Volume Kit (Roche Diagnostics, Almere, the Netherlands) and sequenced on Illumina NextSeq500 (respiratory samples) or Nova-Seq6000 (CSF samples, plasma) instruments using 150 bp paired-end runs after library preparation using New England BioLabs' NEBNext Ultra Directional RNA Library preparation kit for Illumina with in-house adaptations in order to enable simultaneous detection of both DNA and RNA viruses, at the Leiden University Medical Center (LUMC) [4,20]. ...
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Introduction Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories. Methods Metagenomic datasets from 13 clinical samples from patients with encephalitis or viral respiratory infections characterized by PCR were selected. The datasets were analysed with 13 different pipelines currently used in virological diagnostic laboratories of participating ENNGS members. The pipelines and classification tools were: Centrifuge, DAMIAN, DIAMOND, DNASTAR, FEVIR, Genome Detective, Jovian, MetaMIC, MetaMix, One Codex, RIEMS, VirMet, and Taxonomer. Performance, characteristics, clinical use, and user-friendliness of these pipelines were analysed. Results Overall, viral pathogens with high loads were detected by all the evaluated metagenomic pipelines. In contrast, lower abundance pathogens and mixed infections were only detected by 3/13 pipelines, namely DNASTAR, FEVIR, and MetaMix. Overall sensitivity ranged from 80% (10/13) to 100% (13/13 datasets). Overall positive predictive value ranged from 71-100%. The majority of the pipelines classified sequences based on nucleotide similarity (8/13), only a minority used amino acid similarity, and 6 of the 13 pipelines assembled sequences de novo. No clear differences in performance were detected that correlated with these classification approaches. Read counts of target viruses varied between the pipelines over a range of 2-3 log, indicating differences in limit of detection. Conclusion A wide variety of viral metagenomic pipelines is currently used in the participating clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implicating the need for standardization and validation of metagenomic analysis for clinical diagnostic use. Future studies should address the selective effects due to the choice of different reference viral databases.
... A similar pattern of biased U-to-C hypermutation has been described in the context of mumps vaccine virus genomes recovered from the brain of a previously vaccinated pediatric severe combined immunodeficiency (SCID) patient. Consensus hypermutation in the M gene was observed in this strain alongside limited quasispecies diversity by nextgeneration sequencing, suggesting either (i) absence of negative selective pressures acting on divergent M sequences or (ii) positive selection of defective M protein as a means to achieve efficient viral spread in nervous tissue (25). In support of the first mechanism, a similar pattern of biased U-to-C hypermutation in the SH sequence has been observed in nonprimate strains of parainfluenza virus 5, accompanied by mutation of the start codon and loss of SH protein expression (26). ...
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Mumps virus (MuV) outbreaks occur in the United States despite high coverage with measles, mumps, rubella (MMR) vaccine. Routine genotyping of laboratory-confirmed mumps cases has been practiced in the United States since 2006 to enhance mumps surveillance. This study reports the detection of unusual mutations in the small hydrophobic (SH) protein of contemporary laboratory-confirmed mumps cases and is the first to describe the impact of such mutations on SH protein function. These mutations are predicted to profoundly alter the amino acid sequence of the SH protein, which has been shown to antagonize host innate immune responses; however, they were neither associated with defects in virus replication nor attenuated protein function in vitro
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The diagnosis of infectious diseases in immunocompromised hosts presents unique challenges for the clinician. Metagenomic next generation sequencing (mNGS) based diagnostics that identify microbial nucleic acids in clinical samples (mNGS for pathogen identification or mNGSpi) may be a useful tool in addressing some of these challenges. Studies of mNGSpi in immunocompromised hosts have demonstrated that these diagnostics are capable of identifying causative organisms in a subset of patients for whom conventional testing has been negative. While these studies provide proof of concept for mNGSpi utility, they have a number of limitations, which make it difficult to confidently assess test performance and clinical impact based on current data. Future studies will likely feature larger cohort sizes and controlled interventional study designs that assess the impact of mNGSpi on clinical endpoints. They will also likely include assessments of the clinical value of data generated by mNGS beyond pathogen identification.
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Introduction: Meningoencephalitis patients are often severely impaired and benefit from early etiological diagnosis though many cases remain without identified cause. Metagenomics as pathogen agnostic approach can result in additional etiological findings, however the exact diagnostic yield when used as a secondary test remains unknown. Areas covered: This review aims to highlight recent advances with regard to wet and dry lab methodologies of metagenomic testing and technical milestones that have been achieved. A selection of procedures currently applied in accredited diagnostic laboratories is described in more detail to illustrate best practices. Furthermore, a meta-analysis was performed to assess the additional diagnostic yield utilizing metagenomic sequencing in meningoencephalitis patients. Finally, the remaining challenges for successful widespread implementation of metagenomic sequencing for the diagnosis of meningoencephalitis are addressed in a future perspective. Expert opinion: The last decade has shown major advances in technical possibilities for using mNGS in diagnostic settings including cloud-based analysis. An additional advance may be the current established infrastructure of platforms for bioinformatic analysis of SARS-CoV-2, which may assist to pave the way for global use of clinical metagenomics.
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Live attenuated viral vaccines (LAV) have saved millions of lives globally through their capacity to elicit strong, cross-reactive and enduring adaptive immune responses. However, LAV can also act as a Trojan horse to reveal inborn errors of immunity, thereby highlighting important protective elements of the healthy antiviral immune response. In the following article, we draw out these lessons by reviewing the spectrum of LAV-associated disease reported in a variety of inborn errors of immunity. We note the contrast between adaptive disorders, which predispose to both LAV and their wild type counterparts, and defects of innate immunity in which parenterally delivered LAV behave in a particularly threatening manner. Recognition of the underlying pathomechanisms can inform our approach to disease management and vaccination in a wider group of individuals, including those receiving immunomodulators that impact the relevant pathways.
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Background Metagenomic next-generation sequencing offers an unbiased approach to identifying viral pathogens in cerebrospinal fluid of patients with meningoencephalitis of unknown etiology. Methods In an 11-month case series, we investigated the use of cerebrospinal fluid metagenomic next-generation sequencing to diagnose viral infections among pediatric hospitalized patients presenting with encephalitis or meningoencephalitis of unknown etiology. Cerebrospinal fluid from patients with known enterovirus meningitis were included as positive controls. Cerebrospinal fluid from patients with primary intracranial hypertension were included to serve as controls without known infections. Results Cerebrospinal fluid metagenomic next-generation sequencing was performed for 37 patients. Among 27 patients with encephalitis or meningoencephalitis, 4 were later diagnosed with viral encephalitis, 6 had non–central nervous system infections with central nervous system manifestations, 6 had no positive diagnostic tests, and 11 were found to have a noninfectious diagnosis. Metagenomic next-generation sequencing identified West Nile virus (WNV) in the cerebrospinal fluid of 1 immunocompromised patient. Among the 4 patients with known enterovirus meningitis, metagenomic next-generation sequencing correctly identified enteroviruses and characterized the viral genotype. No viral sequences were detected in the cerebrospinal fluid of patients with primary intracranial hypertension. Metagenomic next-generation sequencing also identified sequences of nonpathogenic torque Teno virus in cerebrospinal fluid specimens from 13 patients. Conclusions Our results showed viral detection by cerebrospinal fluid metagenomic next-generation sequencing only in 1 immunocompromised patient and did not offer a diagnostic advantage over conventional testing. Viral phylogenetic characterization by metagenomic next-generation sequencing could be used in epidemiologic investigations of some viral pathogens, such as enteroviruses. The finding of torque Teno viruses in cerebrospinal fluid by metagenomic next-generation sequencing is of unknown significance but may merit further exploration for a possible association with noninfectious central nervous system disorders.
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Type I interferon (IFN-α/β) is a fundamental antiviral defense mechanism. Mouse models have been pivotal to understanding the role of IFN- α /β in immunity, although validation of these findings in humans has been limited. We investigated a previously healthy child with fatal encephalitis after inoculation of the live attenuated measles, mumps, and rubella (MMR) vaccine. By targeted resequencing, we identified a homozygous mutation in the highaffinity IFN- α/β receptor (IFNAR2) in the proband, as well as a newborn sibling, that rendered cells unresponsive to IFN- α /β. Reconstitution of the proband's cells with wild-type IFNAR2 restored IFN- α /β responsiveness and control of IFN-attenuated viruses. Despite the severe outcome of systemic live vaccine challenge, the proband had previously shown no evidence of heightened susceptibility to respiratory viral pathogens. The phenotype of IFNAR2 deficiency, together with similar findings in STAT2-deficient patients, supports an essential but narrow role for IFN- α /β in human antiviral immunity.
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Mumps vaccines are live attenuated viruses. They are known to vary in effectiveness, degree of attenuation and adverse event profile. However, the underlying reasons are poorly understood. We studied two closely related mumps vaccines which originate from the same attenuated Jeryl Lynn-5 strain but have different efficacies. Jeryl Lynn-Canine Kidney (JL-CK), produced on primary canine kidney cells, is less effective than RIT4385, which is produced on chicken embryo fibroblasts. JL-CK and RIT4385 could be distinguished by a number of in vitro and in vivo properties. JL-CK produced heterogeneous, generally smaller plaques than RIT4385, but gave 100-fold higher titres when grown in cells and showed a higher degree of hydrocephalus formation in neonatal rat brains. Sanger sequencing of JL-CK identified 14 regions of heterogeneity throughout the genome. Plaque purification of JL-CK demonstrated the presence of five different Jeryl Lynn-5 variants encompassing the 14 mutations. One JL-CK mutation was associated with a small plaque phenotype, the effects of the others in vitro or in vivo were less clear. Only 4% of the JL-CK population corresponded to the parental Jeryl Lynn-5 strain. Next generation sequencing of JL-CK and virus before and after growth in cell lines or neonatal rat brains showed that propagation in vitro or in vivo altered the population dramatically. Our findings indicate that growth of JL-CK in primary canine kidney cells resulted in the selection of a mixture of mumps virus variants that have different biological properties compared to the parent Jeryl Lynn-5 virus. We also report three previously unknown heterogenic regions within the N gene of the RIT4385 vaccine. Copyright © 2015. Published by Elsevier Ltd.
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Full-text available
Definitive identification of poliovirus strains in vaccines is essential for quality control, particularly where multiple wild-type and Sabin strains are produced in the same facility. Sequence-based identification provides the ultimate in identity testing and would offer several advantages over serological methods. We employed random RT-PCR and high throughput sequencing to recover full-length genome sequences from monovalent and trivalent poliovirus vaccine products at various stages of the manufacturing process. All expected strains were detected in previously characterised products and the method permitted identification of strains comprising as little as 0.1% of sequence reads. Highly similar Mahoney and Sabin 1 strains were readily discriminated on the basis of specific variant positions. Analysis of a product known to contain incorrect strains demonstrated that the method correctly identified the contaminants. Random RT-PCR and shotgun sequencing provided high resolution identification of vaccine components. In addition to the recovery of full-length genome sequences, the method could also be easily adapted to the characterisation of minor variant frequencies and distinction of closely related products on the basis of distinguishing consensus and low frequency polymorphisms. Copyright © 2015. Published by Elsevier Ltd.
Article
Full-text available
Deep sequencing of clinical samples is now an established tool for the detection of infectious pathogens, with direct medical applications. The large amount of data generated produces an opportunity to detect species even at very low levels, provided that computational tools can effectively profile the relevant metagenomic communities. Data interpretation is complicated by the fact that short sequencing reads can match multiple organisms and by the lack of completeness of existing databases, in particular for viral pathogens. Here we present metaMix, a Bayesian mixture model framework for resolving complex metagenomic mixtures. We show that the use of parallel Monte Carlo Markov chains (MCMC) for the exploration of the species space enables the identification of the set of species most likely to contribute to the mixture. We demonstrate the greater accuracy of metaMix compared to relevant methods, particularly for profiling complex communities consisting of several related species. We designed metaMix specifically for the analysis of deep transcriptome sequencing datasets, with a focus on viral pathogen detection, however the principles are generally applicable to all types of metagenomic mixtures. metaMix is implemented as a user friendly R package, freely available on CRAN: http://cran.r-project.org/web/packages/metaMix CONTACT: sofia.morfopoulou.10@ucl.ac.uk SUPPLEMENTARY INFORMATION: Supplementary Material is available at Bionformatics online. © The Author(s) 2015. Published by Oxford University Press.
Article
Full-text available
Background. An 18-month-old boy developed encephalopathy, for which extensive investigation failed to identify an etiology, 6 weeks after stem cell transplant. To exclude a potential infectious cause, we performed high-throughput RNA sequencing on brain biopsy. Methods. RNA-Seq was performed on an Illumina Miseq, generating 20 million paired-end reads. Nonhost data were checked for similarity to known organisms using BLASTx. The full viral genome was sequenced by primer walking. Results. We identified an astrovirus, HAstV-VA1/HMO-C-UK1(a), which was highly divergent from human astrovirus (HAstV 1–8) genotypes, but closely related to VA1/HMO-C astroviruses, including one recovered from a case of fatal encephalitis in an immunosuppressed child. The virus was detected in stool and serum, with highest levels in brain and cerebrospinal fluid (CSF). Immunohistochemistry of the brain biopsy showed positive neuronal staining. A survey of 680 stool and 349 CSF samples identified a related virus in the stool of another immunosuppressed child. Conclusions. The discovery of HAstV-VA1/HMO-C-UK1(a) as the cause of encephalitis in this case provides further evidence that VA1/HMO-C viruses, unlike HAstV 1–8, are neuropathic, particularly in immunocompromised patients, and should be considered in the differential diagnosis of encephalopathy. With a turnaround from sample receipt to result of <1 week, we confirm that RNA-Seq presents a valuable diagnostic tool in unexplained encephalitis.
Conference Paper
Metagenomics can be defined as the study of DNA sequences from environmental or community samples. This is a rapidly progressing field and application ideas that seemed outlandish a few years ago are now routine and familiar. Metagenomics’ scope is broad and includes the analysis of a diverse set of samples such as environmental or clinical samples. Human tissues are in essence metagenomic samples due to the presence of microorganisms, such as bacteria, viruses and fungi in both healthy and diseased individuals. Deep sequencing of clinical samples is now an established tool for pathogen detection, with direct medical applications. The large amount of data generated produces an opportunity to detect species even at very low levels, provided that computational tools can effectively profile the relevant metagenomic communities. Data interpretation is complicated by the fact that short sequencing reads can match multiple organisms and by the lack of completeness of existing databases, particularly for viruses. The research presented in this thesis focuses on using Bayesian Mixture Model techniques to produce taxonomic profiles for metagenomic data. A novel Bayesian mixture model framework for resolving complex metagenomic mixtures is introduced, called metaMix. The use of parallel Monte Carlo Markov chains (MCMC) for the exploration of the species space enables the identification of the set of species most likely to contribute to the mixture. The improved accuracy of metaMix compared to relevant methods is demonstrated, particularly for profiling complex communities consisting of several related species. metaMix was designed specifically for the analysis of deep transcriptome sequencing datasets, with a focus on viral pathogen detection. However, the principles are generally applicable to all types of metagenomic mixtures. metaMix is implemented as a user friendly R package, freely available on CRAN: http://cran.r-project.org/web/packages/metaMix.
Article
Measles virus (MV) is the causative agent of subacute sclerosing panencephalitis (SSPE). We previously reported that the F gene of the SSPE Osaka-2 strain is the major determinant of MV neurovirulence. Because the sites and extents of mutations differ among SSPE strains, it is necessary to determine the mutations responsible for the SSPE-specific phenotypes of individual viral strain. In this study, recombinant viruses containing the envelope-associated genes from the SSPE Osaka-1 strain were generated in the IC323 wild-type MV background. Hamsters inoculated with MV containing the H gene of the Osaka-1 strain displayed hyperactivity and seizures, but usually recovered and survived. Hamsters inoculated with MV containing the F gene of the Osaka-1 strain displayed severe neurologic signs and died. Amino acid substitutions in the heptad repeat A and C regions of the F protein, including a methionine-to-valine substitution at amino acid 94, play major roles in neurovirulence.