The CVN Development Programme a 4-month update

The CVN Development Programme

a 4-month update

Peter Simmonds

Centre for Infectious Diseases University of Edinburgh

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

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

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

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

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)

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

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

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

EV and HPeV transcript standards



Dilution series of 10

– 10

_{RNA copies / ul created for}

each transcript



Aliquoted and stored at -40

_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



RNA expressed quantified by

nanodrop / Agilent



Integrity assessed by

denaturing gel electrophoresis



Storage and dilution

– Citrate buffer, pH6.0 – 0.05 ug/ml carrier tRNA – 0.1 U / ml RNAsin

Real time PCR

examples

CAV16 transcript



Similar results for

other transcripts



Reproducible

between species

Real time PCR

examples

CAV16 transcript



Similar results for

other transcripts



Reproducible

between species

A-D

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) 178-181 (PEV)

Edinburgh

Glasgow

Belfast 1

Sensitivity of reference testing and typing assays

 Nested PCR for 5’UTR  EV serotype identification

– Nested PCR amplification

– sequencing of VP1 (species A, B, D) or VP2 (species C)

Primers RNA 5 x 104 _{5 x 10}3 _{5 x 10}2 _{5 x 10}1 _{5 x 10}0 _{5 x 10}-1 5’UTR A + + + + + -5’UTR B + + + + + -5’UTR C + + + + + -5’UTR D + + + + - -5’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

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

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)

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



– 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