1. Human Parvoviruses Kevin E Brown Immunisation and Diagnosis Unit Virus Reference Department Centre for Infections

2. Parvoviruses Small 22-24 nm non-enveloped 4 - 5.5 kb ss DNA genome with ITRs at 5’ and 3’ ends Encode no polymerase Dependant on cellular/other viral proteins for DNA replication 2 large ORF NS (or Rep) proteins VP (or Cap) proteins Classification Two subfamilies – host range Densovirinae – insects Parvovirinae – vertebrates Autonomous/dependant replication + or – strand packaging Identical or different 5’ and 3’ ITRs 1, 2 or 3 promoters Sequence pyhlogeny

3. Parvovirus B19 First identified in 1974 (CPHL, London) ~ 22 nm icosahedral virion Linear single stranded DNA ~ 5600 nucleotides Long (383nt) terminal hairpin seq Complex transcription map Single promoter (p6) 1 non-spliced non-structural protein 2 capsid proteins (VP1 & VP2) -alt splicing Small proteins of unknown function VP2 self assembles to form VLP Erythrotropic Does not grow in standard tissue culture Replicates in erythroid progenitors Rare cell lines (UT7/Epo; KU812Ep6)

4. Servant et al. J. Virol 2002 Phylogenetic tree of VP1u region V9 A6

5. PanelDatesNoB19 posVariant A HIV patients France 1992-19972111 Type 3 5%Servant, J. Virol 2002 B Fetal hydrops France 1995-1997731600 D B19 Ag positive France 1972-199987 1 Type 3 1% E B19-like symptoms France 1999-2001633889 Type 2 & 3 10% C B19-like symptoms USA 27020400 Blood donors UK 1999-2001100090/4Candotti, J.Virol 2004 Blood donors Ghana 1999-200110001312/12 Type 3 100% B19-like symptoms Brazil 2003-200569127 6 type 3 1 type 2 58%Sanabani, J. Clin Micro 2006 Tissues Finland 52319058* Type 2 11%Norja, PNAS 2006 Prevalence of variant B19 in different groups * Only detected in those born before 1973

6. 1 st identified in a IVDU Fever, arthralgia, meningism Acute sample only Lost to follow up Closest sequence Putative Erythroviruses Chipmunk

7. Parv4 DNA Typical parvovirus features Incomplete hairpin sequence Gap between NS and capsid VP1 ~ 100 kDa No 11 kDa or 7.5kDa ORFs

8. Prevalence of Parv4 DNA by PCR (NIH data) SamplesNHP4-F/R (NS) HP4-2 (Capsid) HP4-3 (Capsid) Clinical samples262013 HAA samples26021 Liver samples17000 Arthropathy200 NT HIV12NT 1 Blood donors20000 Plasma pools62021 German pools6000

9. Blood donors from Los Angeles area summer 2005 300 pools of 16 29 positives (9.6% or 0.6% if divided by 16). 200 single donations 4 positives (2%) Subjects with symptoms of acute viral infection or highly exposed (MSM, IDU) 16/195 subjects positive (85) 1 PARV5, PARV4 Increasing prevalence in symptomatic/exposed subjects may reflect pathogenic viral infection OR higher exposure to blood borne viruses (e.g. like GBV-C) Prevalence of PARV4 virus using nested PCR

10. 10 20 30 40 50 60 70 80 90 100 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ gttgatggccctgtggttagCACCCAGCATCAAGAAGCTTTGCAGACAAGAATAACCATGTTTCAGTTTCAGAGAATGGTTCCGGATGGCTTAGCTCCAC PARV4-Cutter pool-1991........y..................................G........................................................ Poolx16 24/29........y........................................................................................... Poolx16 2/29........y..................................R........................................................ Poolx16 1/29........y........................T.........G........................................................ Poolx16 1/29........y..................................G................................................R....... Poolx16 1/29........y..................................G........................................................ Neat 3/4........y..................................G.....................................T.................. Neat 1/4........y..................................G........................................................ Option 7/16........y........................................................................................... Option 3/16........y........................................................................................... Option 1/16........y..............................................T............................................ Option 1/16........y................R.................G........................................................ Option 1/16........y..................................G...R.................................................... Option 1/16........y.............................C....G........................................................ Option 1/16........y..............T........G......C..................................G........C...........C..T. Option 1/16 --......T..............T........G......C..................................G........C...........C..T. PARV5-Cutter-1991 110 120 130 140 150 160 ---------+---------+---------+---------+---------+---------+- TTCCTGAAGAGGAAGTGAGAAGCTTTTTTAAGCTAGgtgaacaggaactgaatatgaaagg PARV4-Cutter-1991............................................................. Poolx16 24/29............................................................. Poolx16 2/29............................................................. Poolx16 1/29............................................................. Neat 3/4............................................................. Neat 1/4............................................................. Option 7/15............................................................. Option 3/15...............................A............................. Option 1/15............................................................. Option 1/15.C....................T...........T.......................... Option 1/15.C....................T...........T.......................... PARV5-Cutter-1991 Genetic variation among PARV4

11. Parv4 DNA VP2 expression Bacteria 60 kDa Baculovirus VLP No HA ELISA developed Indirect format

12. Seroprevalence of Parv4 <6 months6 months-56-1011-15>16 Washington DC Pos152723289 Equiv26300 Neg77400 Total244030289 Vietnam Pos10129 1 Equiv53210 Neg27210 Total172213141 % Positive73.5%62.9%74.4%95%100%

13. Pooled NPA secretions 48 samples 38/48 pediatric patients 378 NPA samples Culture negative 7/266 pediatric patients 0/112 adult patients 540 samples hospitalized peds 3.1% pos Increased in winter months Closest sequence Bovine parvovirus CPV type 1 (MVC)

14. Human parvoviruses Parvovirus B19 (B19V) – type member of Erythroviruses 3 different genotypes now recognized No evidence for different serotypes Highly erythrotropic Detection of DNA in blood and tissues for years following acute infection No evidence of reactivation/integration Parv4/5 is a new member of the Parvovidae Sequences are uncommon in blood and blood products No culture method available VLP can be expressed Do not resemble B19V Do not HA Parv4 Ab common in children/adults Human bocavirus in respiratory secretions of children < 5 Prevalence in other tissues/age groups VLP can be expressed Adeno-associated viruses – AAV2, 3, 5 are human viruses

15. Acknowledgements Virus Discovery Group Jun Lu Ian Mills Tri Nguyen Susan Wong Ning Zhi Hematology Branch Neal Young Sachiko Kajigaya Outside collaborations Mavis Agbanje-McKenna Sally Baylis Eric Delwart Erik Heegaard Mei-Ying Yu Health Protection Agency Hazel Appleton