NDV Technical roundup:

Update on NDV/APMV-1 pathotyping assay

(“Pathstat”) and testing algorithm for APMV-1

David Sutton, Brandon Löndt & Ian Brown

22nd Joint Annual Meetings of the National Reference

Laboratories for Avian Influenza and Newcastle Disease

Copenhagen 18-21 April 2016

Original Collins/Aldous method

~780bp

374bp for phylogenetic analyses

NDV conventional RT-PCR re-design

• Improved sensitivity

• Potential for enhanced turnaround times in NDV detection

• * alignment to consistent approach for APMV-1 phylogenetic analyses

200bp for F cleavage site

Full F gene for phylogenetic analyses*

Specificity • Panel of contemporary isolates comprising

57 APMV-1 isolates covering all lineages* and regions

18 negative - 1 each of APMV-2 to 9, 1 each of AmPV-A to C, 3 x

IBV & 4 x AIV

RNA extracted from cultured virus

positives tested from 1:100 dilution, non-APMV1 virus tested

undiluted

200bp for F cleavage site

Sequence data for 22 isolates confirmed that

Pathstat primers matched sequences derived from

original primer set

Test To Exclude (TTE) scheme

• Clinical samples - swabs/tissues

Fuller 2009

New “Pathstat”

Original and Pathstat primers on BPL

inactivated lyophilized EU panel samples • Sample 2 and 6 – strong and

weak titre of a mixed AI/NDV

isolate (Israel lin. 5d)

- Existing primers fail with

weaker dilution

- Pathstat primers (proposed

new) amplify both strong and

weaker dilutions

• Sample 9 NDV isolate

(Romania 5d)

- Failure with existing primers

- Pathstat primers success

• Lineage 6 is lineage 6 specific

forward pathstat primer

Pathstat primers 200bp product

Original primers 780bp productl

Case 3

Current status and proposed approaches for

APMV1 diagnosis

Molecular Classical

Screening: MGB L gene Isolation in Eggs/Cells

Virulence: ‘Pathotyping’ F PCR HA /antibody typing

Pathostat

avirulent?

ICPI

Green font ISO17025 acceditation in application at EURL

Black font ISO17025 acceditation at EURL

Acknowledgements

• Scott Reid

• Steve Essen