The CVN Development Programme a 4-month update

Transcription

1 The CVN Development Programme a 4-month update Peter Simmonds Centre for Infectious Diseases University of Edinburgh Edinburgh

2 CVN Development Programme Initiative announced in 2009 to focus development towards defined diagnostic issues: Ascribing quantitative values to molecular assay controls Calibration of NIBSC / SoGAT international standards into molecular copies Coordination with NIBSC / CVN quality control Standardisation of EBV, HCMV and adenovirus quantitation Predictive value of quantitative assays Choice of specimen type Applicants Development programme Viral diversity and effect on diagnostic assay sensitivity Role of parechoviruses and enteroviruses in severe neonatal disease Development of bioinformatic and virus sequence database resources

3 CVN Development Programme Viral diversity and effect on diagnostic assay sensitivity Species (ie. enteroviruses, rhinoviruses), genogroups (eg. Norovirus) and genotypes (eg. RSV, hmpv) Viruses that have drifted away from reference isolate sequences (eg. PIVs) Role of parechoviruses and enteroviruses in severe neonatal disease Development of more sensitive and effective assays for CSF screening, protocols of obtaining other sample types EV and HPeV virus typing investigation of specifically neurovirulent types (eg. HPeV3) Establishment of effective routine (sero)type identification methods for UK-wide surveillance Development of bioinformatic and virus sequence database resources Large-scale, updated sequence alignments Software development and interfacing

4 CVN Development Programme Viral diversity and effect on diagnostic assay sensitivity Species (ie. enteroviruses, rhinoviruses), genogroups (eg. Norovirus) and genotypes (eg. RSV, hmpv) Viruses that have drifted away from reference isolate sequences (eg. PIVs) Role of parechoviruses and enteroviruses in severe neonatal disease Development of more sensitive and effective assays for CSF screening, protocols of obtaining other sample types EV and HPeV virus typing investigation of specifically neurovirulent types (eg. HPeV3) Establishment of rapid turnaround, effective routine (sero)type identification methods for UK-wide surveillance Development of bioinformatic and virus sequence database resources Large-scale, updated sequence alignments Software development and interfacing

5 CVN Development Programme Viral diversity and effect on diagnostic assay sensitivity Species (ie. enteroviruses, rhinoviruses), genogroups (eg. Norovirus) and genotypes (eg. RSV, hmpv) Viruses that have drifted away from reference isolate sequences (eg. PIVs) Role of parechoviruses and enteroviruses in severe neonatal disease Development of more sensitive and effective assays for CSF screening, protocols of obtaining other sample types EV and HPeV virus typing investigation of specifically neurovirulent types (eg. HPeV3) Establishment of rapid turnaround, effective routine (sero)type identification methods for UK-wide surveillance Development of bioinformatic and virus sequence database resources Large-scale, updated sequence alignments Software development and interfacing

6 Picornaviruses Positive stranded RNA viruses Primarily infect mammals Capable of acute resolving or persistent infections Highly variable host ranges, disease associations and epidemiologies Conserved design and replication strategy 14 genera identified to date, more to come! No clear idea of the time frame over which different groups (genera, species, serotypes evolved)

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8 Enterovirus Species The Enterovirus genus classified into: 5 enterovirus species, A-E 3 rhinovirus species, A-C 2 simian species, A, B 2 bovine species, A, B 1 porcine species Differ substantially from each other through genome (>35%) Shared 5 UTRs through recombination Each species comprises a variable number of serologically distinct serotypes Identifiable by cross-neutralisation assay 93 EV serotypes, 160 HRV types Only sequences from the capsid region identify serotype

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10 Enterovirus diagnosis EVs readily isolatable in a variety of cell lines Standard method for EV detection until molecular tests developed Serotype identification by cross-neutralisation Isolation more effective for certain serotypes and species (eg. species B) Time-consuming, serotype identification technically difficult (increasing number of types identified) Enterovirus molecular diagnostics Highly conserved 5 UTR typically targeted by PCR-based screening assays Highly sensitive, eg. much more effective detection of EV RNA in CSF Screening equally effective for different species greater role of species A in clinical presentations than recognised previously Enterovirus typing 5 UTR sequence alone is unable to identify species or serotype VP1 sequences allow accurate type identification Reliable typing assays problematic to develop because of variability in target region

11 EV and HPeV transcript standards Investigation of the effectiveness of screening assays for different EV species Analytical sensitivity, required for CSF screening Interpretation of late rises in Ct value Cross-reactivity with human rhinoviruses (with similar 5 UTR sequence) Effectiveness of typing assays for each species Full length transcripts to allow assays targeting different regions to be evaluated Species A: CVA16 Species B: Echo7, Echo30 Species C: CVA21 Species D: EV70 HRV species A (1B) and B (14)

12 EV and HPeV transcript standards RNA expressed quantified by nanodrop / Agilent Integrity assessed by denaturing gel electrophoresis Storage and dilution Citrate buffer, ph ug/ml carrier trna 0.1 U / ml RNAsin Dilution series of RNA copies / ul created for each transcript Aliquoted and stored at -40 o C Used to evaluate sensitivity of RIE diagnostic PCR EV/HPeV multiplexed PCR EV species A, B, C and D VP1 primers used for virus typing

13 Real time PCR examples CAV16 transcript Replicate testing from 9 x 10 5 to 9 x 10-1 RNA copies Positive down to 90 copies Similar results for other transcripts Reproducible between species

14 Real time PCR examples CAV16 transcript Replicate testing from 9 x 10 5 to 9 x 10-1 RNA copies Positive down to 90 copies Similar results for other transcripts Reproducible between species A- D

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24 Inter-lab variability Glasgow RVL

25 Inter-lab variability Belfast Method 1 * Method 2 ** * H O Neill (EV) and Benschop K, Beld M et al JCV 41 (2008) 69-74; Oberste MS et al JMV 58 (1999) (PEV) **Heim A et al JCV 42 (2008) (EV)

26 Edinburgh Belfast 1 Glasgow Belfast 2

27 Sensitivity of reference testing and typing assays Primers RNA 5 x x x x x x UTR A UTR B UTR C UTR D UTR HRV-B Sp. A A 4/4 4/4 0 0 Sp. B B 4/4 1/4 0 0 Sp. C C 4/4 2/4 2/4 0 Sp. D D 4/4 3/4 1/4 0 Nested PCR for 5 UTR EV serotype identification Nested PCR amplification sequencing of VP1 (species A, B, D) or VP2 (species C)

28 EV, HRV and HPeV standards EV species variability Little or no effect on 5 UTR based PCR Equivalent amplification dynamics and sensitivity 2-9 copy control produces Ct values >36, frequently low, interpretation difficulties HPeV detection Highly comparable to EV detection No cross-reactivity between assays HRV detection Standard detection of HRV-1B (species A) Approximately 10-fold reduction in amplification efficiency for HRV-14 (species B) Likely caused by terminal base mismatch in sense primer Assay evaluation of EV, HPeV and HRV screening HRV-C transcript to be added Reference laboratory testing Distribution to diagnostic labs collation of sensitivity data

29 Respiratory viruses Representation of controls generated from viruses currently circulating the UK Full length clones inappropriate / unavailable Whole gene transcripts from major respiratory viruses: RSV genotypes A and B* hmpv genotypes A and B* Influenza A H1N1*, H3N2**, H5N1 and sh1n1** Influenza B (x 3)** Parainfluenza viruses 1-3 (x 2 each)** Variability in regions targeted by diagnostic PCR RSV and hmpv: Nucleoprotein IFA: Matrix, IFB: NP PIV 1-3: HN protein Second round of transcripts from alternative gene targets Future Plans Additional respiratory viruses Extension to enteric viruses later in the year *Available now (contact Peter Simmonds / Nigel McLeish) **Available early March

30 Thanks to Virus Evolution Group, Centre for Infectious Diseases Nigel McLeish, Carol Leitch, Jeroen Witteveldt, Elly Gaunt Specialist Virology Laboratory, Royal Infirmary of Edinburgh Heli Harvala, Kate Templeton Ingo Johannessen Regional Virology Laboratory, Glasgow Susan Bennett, Rory Gunson Virology Laboratory, Belfast Susan Feeney, Alison Watt, Peter Coyle EV and HPeV Molecular Clones Biological Sciences, University of Warwick, David J. Evans Biological Sciences, University of Essex Glyn Stanway Funding UK Clinical Virology Network