Detection of parvovirus B19 and novel human parvoviruses in highrisk individuals

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Detection of parvovirus B19 and novel human
parvoviruses in high-risk individuals

• Ashleigh Manning1, Kate Templeton2,
• Ed. Gomperts3, Peter Simmonds1,2
• 1Centre for Infectious Diseases, University of
  Edinburgh
• 2Specialist Virology Laboratory, Royal Infirmary
  of Edinburgh
• 3Hospital for Sick Children, Los Angeles

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Human parvovirus infections

• Human parvovirus B19
• Widespread in human populations
• 3 genotypes, limited genetic heterogeneity
• Acute, resolving infections, associated with
  intense viraemia
• Recent evidence for long term persistence
• 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
• 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)

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Novel Parvoviruses in humans

• Parvoviridae
• Wide range of diverse viruses infecting mammals
• Highly host-specific
• Acute resolving infections
• Highly transmissible, stable in environment
• Human Parvoviruses
• Human Erythrovirus (B19)
• PARV4 (Jones et al., 2005)
• Acute infection syndrome
• Little known about epidemiology
• Human Bocavirus (Allander et al., 2005)

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Study Aims

• Human Growth and Development Cohort
• 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 gt 10 years
• Subject to several clinically-based and
  virological natural history studies
• Edinburgh Respiratory Archive
• LREC approval for construction of anonymous
  archives
• Clinical and epidemiological information
  recorded, incapable of identifying specific
  patient
• Sample type and month, donor code, age band,
  location codes,
• Supplied clinical information, Results of other
  diagnostic tests (viral and bacteriological)

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Study Methods

• PCR-based Parvovirus Detection
• Highly conserved region identified in NS
• Nested PCR with B19, PARV4 and HBoV-specific
  primers
• Calibration and Run Controls
• NIBSC Run control, calibrated to B19
  International Standard
• Quantified plasma samples containing PARV4
  variant, PARV5
• Cloned, full length pre-quantified HBoV plasmid
• All assays detected single copies of target
  sequence
• Virus Screening
• Nucleic acid extracted by Qiagen MinElute
• 50 ul effective test volume for plasma

Primers 1000 100 10 1 0.1 Neg PARV4(5)
8/8 16/16 16/16 8/16 1/16 0/8
HBoV 12/12 12/12 12/12 5/12 1/12 0/25
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Haemophilia Screen

• Single samples from 59 haemophiliacs
• Test sensitivity 20 DNA copies / ml
• All sample negative for B19 and HBoV

Primers Positive Tested
Frequency Parvovirus B19 0 59
0 Human Bocavirus 0 59
0 PARV4/5 2 59 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
• One identical to PARV4 over 216 bases
• One showed 14 substitutions, all synonymous (6.5)

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Respiratory Screen

• 942 respiratory samples from 589 individuals
• Human Bocavirus
• 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
• Parvovirus B19
• 4 positive from 3 individuals for B19
• 1 persistent infection in an immunosuppressed
  adult
• PARV4/5
• All samples negative

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HBoV Epidemiology

• Closely resembles RSV in epidemiology
• Peak incidence December/January
• Infections largely confined to lt 2 years of age
• Strongly associated with lower respiratory tract
  infections
• Frequent HBoV / RSV or adenovirus coinfections
• Potential exacerbating role in LRTIs

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Detection of parvovirus B19 and novel human
parvoviruses in high-risk individuals
Centre for Infectious Diseases, University of
Edinburgh Ashleigh Manning Peter Simmonds
Sick Childrens Hospital Los Angeles Ed Gomperts
ACKNOWLEDGEMENTS HGDS CoordinatingGroup Sally
Baylis Tobias Allander

• Specialist Virology Laboratory, Royal Infirmary
  of Edinburgh
• Kate Templeton