Boost Fertility New Invention Ups Success Rates.pdf
Sonal 201113 davos
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.
Probes
Solid Base
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
Avian viruses covered by the array
Virus family
Herpesviridae
Astroviridae
Poxviridae
Bornaviridae
Virus groups
Alphaherpesvirus
Astrovirus
Avipox virus
Borna disease virus
Circovirus
Circoviridae
Gyrovirus
Coronaviridae
Coronavirus
Togaviridae
Eastern equine
encephalitis virus
Birnaviridae
Gumboro disease virus
Orthomyxoviridae Influenza A virus
Metapneumovirus
Paramyxoviridae
Paramyxovirus 1-12
Parvoviridae
Parvovirus
Picornavirus
Picornaviridae
Duck Hepatitis A Virus
Polyomaviridae
Polyomavirus
Virus family
Reoviridae
Togaviridae
Virus groups
Reovirus
Sindbis virus
Flavivirus (other)
Japanese encephalitis virus
Murray Valley encephalitis virus
Flaviviridae
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
7. Material & Methods
Sample
Preparation
Hybridisation
Array Imaging
and Analysis
~ 8 – 10hrs
Unique combination
of random
amplification &
specific biotin
labelling (adapted
from Gurrala et al.,
2009)
The identibac hybridisation
kit (Alere Technologies)
The ArrayMate (Alere
Technologies)
IconoClust software (Alere
Technologies)
R script analysis
8. Sensitivity Testing
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.
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. Array verification using known
virus samples
Virus Isolates
aPMV
No. of
strains
10
Showed clear
signal
Known Clinical
samples
10
No. of
Showed
sample clear
signal
Astrovirus (Turkey)
2
2
IBV & Astrovirus
(Turkey)
1
1
IBV (Turkey)
1
1
IBV
3
Not detected
Influenza A
9
9
aMPV
3
3
Parvovirus (Goose)
1
1
Reovirus
1
1
Reovirus (Turkey)
2
1
Sindbis virus
1
1
WNV (Magpie, Greece)
1
Not detected
Kunjin virus
1
1
LIV
1
1
TBEV
1
1
Usutu
1
1
WNV
3
3
10. Disease Investigation
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
1
1 x Reovirus
Swans were found dead in rearing
pens around July 2012 showing
intestinal deformities.
Unknown Clinical
samples
isolate)
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
• All confirmatory PCRs tests
negative
Swedish
Adenovirus
Greek
2
0
6
aMPV
Confirmatory PCR
0
aPMV
1
0
Astrovirus
4
0
4
Circovirus
0
Coronavirus
1
0
Dependovirus
3
-
Sample Quality
Flavivirus
Found dead
0
Hepatitis A
Hunted
4
5
-
Hepatitis B+E
Sindbis
3
1
-
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
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
Funding:
Bioinformatics:
Javier Nunez-Garcia
Other AHVLA members:
Falko Steinbach
Jackie Fenner
Muriel Mafura
MVIU
Nikki MacLaren
Roderick Card
Sahar Mahmood
Sarah McGowan
VI5 Students
