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Development and application of a mini DNA microarray for the screening of wild bird populations in Europe for viral pathogens
1. Development and application of a mini DNA microarray for
the screening of wild bird populations in Europe for viral
pathogens
Sonal Shah
2. Background
Sources of Disease
• Animals are a major source of disease with 75% of all infectious
diseases originating from wildlife over the last few decades.
• Approximately 61% of identified human pathogens are zoonotic,
transferred directly or following mutations.
• Growth in the global population and migration of humans and animals
around the world has increased prevalence of new and emerging
pathogens in animals.
• To allow effective management of future disease threats it is vital to
monitor this large reservoir for infectious pathogens.
3. Background
Current Gold Standard is PCR based
• Rapid
• Sensitive
• Low Cost
• Ideal for known or suspect cases
Potential problems with PCR
• Very specific, may not detect emerging genotypes
• Difficult to multiplex
• Not suitable for unknown cases
4. Background
• DNA microarray consist of a collection
of hundreds of microscopic DNA spots
attached to a solid surface (glass or
silicone).
• Each DNA spot is composed of a
specific DNA sequence, known as
probes or oligonucleotides.
• There are many different types of
microarray platforms available.
Solid Base
Probes
5. Avian Array Features
Consists of approximately 600 probes designed on the available
conserved genomic regions of avian viruses.
Covers a broad range of avian viruses.
The array is printed in a strip format (Alere Technologies), each consisting
of 8 individual arrays.
6. Avian Array Features
Virus family Virus groups
Herpesviridae Alphaherpesvirus
Astroviridae Astrovirus
Poxviridae Avipox virus
Bornaviridae Borna disease virus
Circoviridae
Circovirus
Gyrovirus
Coronaviridae Coronavirus
Togaviridae Eastern equine
encephalitis virus
Birnaviridae Gumboro disease virus
Orthomyxoviridae Influenza A virus
Paramyxoviridae
Metapneumovirus
Paramyxovirus 1-12
Parvoviridae Parvovirus
Picornaviridae
Picornavirus
Duck Hepatitis A Virus
Polyomaviridae Polyomavirus
Virus family Virus groups
Reoviridae Reovirus
Togaviridae Sindbis virus
Flaviviridae
Flavivirus (other)
Japanese encephalitis virus
Murray Valley encephalitis virus
St. Louis encephalitis virus
Usutu virus
West Nile virus
Tick Borne encephalitis virus
Louping ill virus
Adenoviridae Adenovirus
Hepeviridae Avian Hepatitis E Virus
Hepadnaviridae Duck Hepatitis B Virus
Avian viruses covered by the array
7. Material & Methods
Unique combination
of random
amplification &
specific biotin
labelling (adapted
from Gurrala et al.,
2009)
Hybridisation Array Imaging
and Analysis
Sample
Preparation
The identibac hybridisation
kit (Alere Technologies)
The ArrayMate (Alere
Technologies)
IconoClust software (Alere
Technologies)
R script analysis
~ 8 – 10hrs
8. Nucleic acid (NA) extracted from a
known clinical avian parvovirus
sample was serially diluted.
To determine detection limit of avian
parvovirus specific PCR, NA
dilutions were PCR amplified and
visualised on a 2 % agarose gel.
Sensitivity Testing
Ducks experimentally infected with
Avian Influenza A were also used to
determine the limit of array detection.
Nucleic acid was extracted from
cloacal swabs and viral load was
determined using a reverse
transcriptase qPCR (M gene) before
being tested on the array.
9. Virus Isolates No. of
strains
Showed clear
signal
aPMV 10 10
IBV 3 Not detected
Influenza A 9 9
aMPV 3 3
Reovirus 1 1
Sindbis virus 1 1
Kunjin virus 1 1
LIV 1 1
TBEV 1 1
Usutu 1 1
WNV 3 3
Known Clinical
samples
No. of
sample
Showed
clear
signal
Astrovirus (Turkey) 2 2
IBV & Astrovirus
(Turkey)
1 1
IBV (Turkey) 1 1
Parvovirus (Goose) 1 1
Reovirus (Turkey) 2 1
WNV (Magpie, Greece) 1 Not detected
Array verification using known
virus samples
10. Disease Investigation
Unknown Clinical
samples
No. of
samples
Showed clear
signal
Falcon samples 2 1x Circovirus
Manx Sheerwater 4 No virus detected
Flamingo 1 No virus detected
Magpie London 1 No virus detected
Swan 6 6 x Reovirus
Swan (suspect reovirus
isolate)
1 1 x Reovirus
Swans were found dead in rearing
pens around July 2012 showing
intestinal deformities.
EM was initially used to identify
reovirus in the virus culture.
For confirmation, a pan reovirus RT-
PCR for the L2 segment (polymerase
gene, Wellehan Jr et al, 2009) and
sequencing were undertaken for all
swan samples.
Phylogenetic analysis, based on 36
amino acids of L2 segment, revealed
two different strains of ARV in the
affected swans.
12. Avian Reovirus
Reovirus genome consists of a 23.5
Kb double-stranded segmented RNA.
ARVs are associated with a wide
range of disease syndromes in
commercial chickens and turkeys.
Transmitted horizontally by faecal
oral route and contaminated egg
shells and vertically from infected
hens to their chicks.
Increase in cases of ARVs being
reported from a wide range of avian
species.
Recent studies describe new isolates
from broilers that differ from the
classical strains used in commercial
vaccines.
13. Surveillance
• Swedish common eider (x42)
– Reproductive failure
– 33 hunted & 9 found dead
• Greek corvids (x 16)
– No clinical history provided
– All hunted
Swedish Greek Confirmatory PCR
Adenovirus 2 0
aMPV 6 0
aPMV 1 0
Astrovirus 4 0
Circovirus 4 0
Coronavirus 1 0
Dependovirus 3 -
Flavivirus 4 0
Hepatitis A 5 -
Hepatitis B+E 3 -
Sindbis 1 -
Sample Quality
Hunted Found dead
• All confirmatory PCRs tests
negative
14. Conclusions
Sensitivity testing indicates the array is 100-fold less sensitive compared to the
conventional PCR.
In terms of virus genome, the array could detect down to 1.7x 102 virus
genomes from the Influenza A samples.
The array has proven its potential as a frontline tool in the investigation of
suspected avian viral disease syndromes, supported by detection of highly
pathogenic IBV (turkey) and novel ARVs (swans).
Phylogenetic analysis of swan reoviruses revealed two genetically diverse
strains of the virus.
The low cost, ease of use, and short turnaround time provides a desirable
multiplex assay for a broader user base compared to other microarrays of its
type.
15. Acknowledgments
Funding:
Supervisors:
Akbar Dastjerdi (AHVLA)
Paul Barrow (University of Nottingham)
Co- Supervisors:
Liljana Petrovska (AHVLA)
Abu-Bakr A. K. Abu-Median (University of Nottingham)
Provision of virus strains:
Chad Fuller
Charalambos Billinis
Dan Horton
Dolores Gavier-Widén
Elizabeth Aldous
Karen Mansfield
Marek Slomka
Nick Johnson
Scott Reid
Bioinformatics:
Javier Nunez-Garcia
Other AHVLA members:
Falko Steinbach
Jackie Fenner
Muriel Mafura
MVIU
Nikki MacLaren
Roderick Card
Sahar Mahmood
Sarah McGowan
VI5 Students