Transcript Development and Evaluation of a Highly Sensitive, Multiplexed Real

Detection of parvovirus B19 and novel human
parvoviruses in high-risk individuals
Ashleigh Manning1, Kate Templeton2,
Ed. Gomperts3, Peter Simmonds1,2
1Centre
2Specialist
for Infectious Diseases, University of Edinburgh
Virology Laboratory, Royal Infirmary of Edinburgh
3Hospital for Sick Children, Los Angeles
Human parvovirus infections
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Human parvovirus B19
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Widespread in human populations
3 genotypes, limited genetic heterogeneity
Acute, resolving infections, associated with intense viraemia
Recent evidence for long term persistence
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Frequent detection in autopsy tissue, despite lack of persistent
viraemia
Strong, life-long CTL reactivity to B19 antigens, suggests
ongoing low-level replication
Novel human parvoviruses
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Genome-based Virus discovery
Based on molecular methods to detect non-host DNA or RNA
sequences within samples
Recent description of novel human parvoviruses (Allander et
al., 2005; Jones et al., 2005)
Novel Parvoviruses
in humans
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Parvoviridae
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Wide range of diverse viruses
infecting mammals
Highly host-specific
Acute resolving infections
Highly transmissible, stable in
environment
Human Parvoviruses
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Human Erythrovirus (B19)
PARV4 (Jones et al., 2005)
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Acute infection syndrome
Little known about epidemiology
Human Bocavirus
(Allander et al., 2005)
Study Aims
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Human Growth and Development Cohort
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NIH-supported prospectively collected cohort
Recipients of non-virally inactivated factor VIII and IX
concentrates
6-monthly assessment and sample archiving.
Prospectively collected samples for > 10 years
Subject to several clinically-based and virological natural history
studies
Edinburgh Respiratory Archive
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LREC approval for construction of anonymous archives
Clinical and epidemiological information recorded, incapable of
identifying specific patient
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Sample type and month, donor code, age band, location codes,
Supplied clinical information, Results of other diagnostic tests (viral
and bacteriological)
Study Methods
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PCR-based Parvovirus Detection
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Highly conserved region identified in NS
Nested PCR with B19, PARV4 and HBoV-specific primers
Calibration and Run Controls
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NIBSC Run control, calibrated to B19 International Standard
Quantified plasma samples containing PARV4 variant, PARV5
Cloned, full length pre-quantified HBoV plasmid
Primers
PARV4(5)
HBoV
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1000
8/8
12/12
100
16/16
12/12
10
16/16
12/12
1
8/16
5/12
0.1
1/16
1/12
Neg
0/8
0/25
All assays detected single copies of target sequence
Virus Screening
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Nucleic acid extracted by Qiagen MinElute
50 ul effective test volume for plasma
Haemophilia Screen
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Single samples from 59 haemophiliacs
Test sensitivity 20 DNA copies / ml
All sample negative for B19 and HBoV
Primers
Positive
Parvovirus B19
0
Human Bocavirus
0
PARV4/5
2
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Tested
59
59
59
Frequency
0%
0%
3.4%
Two samples positive for PARV4/5
One haemophiliac HIV+/HCV+, one HCV+ only
Relatively high viral load, positive in 1st round
Genetic characterisation
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One identical to PARV4 over 216 bases
One showed 14 substitutions, all synonymous (6.5%)
Respiratory Screen
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942 respiratory samples from 589 individuals
Human Bocavirus
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Parvovirus B19
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53 positive from 37 individuals for HBoV
Almost invariably non-persistent, short period of excretion
Generally confined to infants and young children
Three adults with immunosuppression (transplant) showed
persistent infections (2 from 3 with multiple samples), high
titres
4 positive from 3 individuals for B19
1 persistent infection in an immunosuppressed adult
PARV4/5
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All samples negative
HBoV Epidemiology
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Closely resembles RSV in epidemiology
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Peak incidence December/January
Infections largely confined to < 2 years of age
Strongly associated with lower respiratory tract infections
Frequent HBoV / RSV or adenovirus coinfections
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Potential exacerbating role in LRTIs
Detection of parvovirus B19 and novel human
parvoviruses in high-risk individuals
Centre for Infectious
Diseases, University of
Edinburgh
Ashleigh Manning
Peter Simmonds
Specialist Virology
Laboratory, Royal Infirmary
of Edinburgh
Kate Templeton
Sick Children’s Hospital
Los Angeles
Ed Gomperts
ACKNOWLEDGEMENTS
HGDS CoordinatingGroup
Sally Baylis
Tobias Allander