1. Detection of parvovirus B19 and novel human parvoviruses in high-risk individuals Ashleigh Manning 1, Kate Templeton 2, Ed. Gomperts 3, Peter Simmonds 1,2 1 Centre for Infectious Diseases, University of Edinburgh 2 Specialist Virology Laboratory, Royal Infirmary of Edinburgh 3 Hospital for Sick Children, Los Angeles

2. 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)

3. 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)

4. 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 > 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)

5. 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 Primers1000100 10 10.1Neg PARV4(5) 8/816/1616/168/161/16 0/8 HBoV12/1212/1212/125/121/120/25

6. Haemophilia Screen Single samples from 59 haemophiliacs Test sensitivity 20 DNA copies / ml All sample negative for B19 and HBoV Primers PositiveTested Frequency Parvovirus B19 0 59 0% Human Bocavirus0 59 0% PARV4/52 59 3.4% Two samples positive for PARV4/5 One haemophiliac HIV+/HCV+, one HCV+ only Relatively high viral load, positive in 1 st round Genetic characterisation One identical to PARV4 over 216 bases One showed 14 substitutions, all synonymous (6.5%)

7. 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

8. HBoV Epidemiology Closely resembles RSV in epidemiology 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 Potential exacerbating role in LRTIs

9. Specialist Virology Laboratory, Royal Infirmary of Edinburgh Kate Templeton Centre for Infectious Diseases, University of Edinburgh Ashleigh Manning Peter Simmonds Detection of parvovirus B19 and novel human parvoviruses in high-risk individuals Sick Children’s Hospital Los Angeles Ed Gomperts ACKNOWLEDGEMENTS HGDS CoordinatingGroup Sally Baylis Tobias Allander