Sonal 201113 davos
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Sonal 201113 davos Presentation Transcript

  • 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.
  • 11. Avian Reovirus (ARV) characterisation 56 0.05 Avian_ABF82230.1 Gallus_AEZ06573.1 AEZ06574.1 Turkey_AV00085-AVP-12-002428 ACH72476.1 Broiler_YP_004226522.1 66 ACH72475.1 Goose_orthoreovirus_AFQ62079.1 Swan_8636 Swan_8322 Swan_8721 36 Swan_8634 Swan_8321 Swan_8317 Swan_8633 Duck_reovirus_AFV52258.1 AFV52270.1 80 Duck_reovirus_AGJ98430.1 AGH25586.1 Swan isolate Steller_sea_lion_AED99918.1 61 Magpie London 56 AEQ49381.1 58 AEQ49363.1 99 Bat_YP_007507325.1 Bat_AEQ49375.1 AGI97912.1 AFQ41025.1 Mammalian_ABP48913.1 AFN01893.1 99 ADY80532.1 AFQ37937.1 ADY80522.1 63 ADY16666.1 ADD11993.1 Human_ACZ53985.1 AAL36027.1 Masked_palm_civet_YP_003199418.1 ABG49449.1 Grass_carp_AGG53846.1
  • 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