Multiplex analysis of avian respiratory viruses

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1 Multiplex analysis of avian respiratory viruses Gordon Kirby () & Davor Oykitch (AHL)

2 Animal Health Laboratory U of Guelph OMAFRA Practitioners & Producers Public Health Other Universities CFIA

3 AHL Caseload Distribution ~65,000 cases, over 1,000,000 tests

4 Virus Detection: Isolation in Eggs Influenza A virus Avian paramyxovirus-1 Herpesvirus Adenovirus

5 Virus Detection: Cell Culture Uninfected 24 hrs post-infection

6 Polymerase Chain Reaction (PCR) AGENT FAMILY GENUS Avian adeno-associated virus Parvoviridae Dependovirus AAdV Avian bornavirus Bornaviridae Bornavirus ABV Avian encephalomyelitis virus Picornaviridae Tremovirus AEV Avian nephritis virus Astroviridae Avastrovirus ANV Avian paramyxovirus-1 Paramyxoviridae Avulavirus APMV-1_M Avian paramyxovirus-1 Paramyxoviridae Avulavirus APMV-1_F Avian reovirus Reoviridae Orthoreovirus ARV Chicken anemia virus Circoviridae Gyrovirus CAV Chicken astrovirus 1 Astroviridae Avastrovirus CAstV-1 Duck adenovirus 1 Adenoviridae Atadenovirus DAdV-1 Fowl adenovirus Adenoviridae Aviadenovirus FAdV Infectious bronchitis virus Coronaviridae Coronavirus IBV Infectious bursal disease virus Birnaviridae Avibirnavirus IBDV Infectious laryngotracheitis virus Herpesviridae Iltovirus ILTV Equine eastern encephalitis virus Togaviridae Alphavirus EEEV Influenza A virus Orthomxyoviridae Influenza A Infl A_M Influenza A virus Orthomxyoviridae Influenza A Infl A_H5 Influenza A virus Orthomxyoviridae Influenza A Infl A_H7 Influenza A virus Orthomxyoviridae Influenza A Infl A_H3N2 Influenza A virus Orthomxyoviridae Influenza A Infl A_H1N1 Poxvirus Poxviridae Avipoxvirus Pox West Nile virus Flaviviridae Flavivirus WNV

7 Syndromic testing Respiratory Influenza A virus Avian paramyxovirus 1 Infectious laryngotracheitis virus Infectious bronchitis virus Fowl adenovirus Avian reovirus Poxvirus Enteric Avian astrovirus Avian nephritis virus Avian reovirus Avian rotavirus Chicken anemia virus Fowl adenovirus Infectious bursal disease virus

8 Syndromic testing Respiratory Influenza A virus matrix Influenza H5 Influenza H7 Avian paramyxovirus-1 matrix APMV1- fusion Infectious laryngotracheitis virus Infectious bronchitis virus Enteric Avian astrovirus Avian nephritis virus Avian reovirus Avian rotavirus Chicken anemia virus Fowl adenovirus Infectious bursal disease virus

Development of Avian Respiratory Viral Panel Control of respiratory tract infections is essential due to: high flock morbidity and mortality significant economic damage Speed and accuracy of

9 Development of Avian Respiratory Viral Panel Control of respiratory tract infections is essential due to: high flock morbidity and mortality significant economic damage Speed and accuracy of diagnosis reduces: burden of disease, facilitates disease monitoring, prevents unnecessary flock eradication minimizes public health risks New analytical methods that screen for multiple respiratory viral pathogens of poultry are need

10 Method Development Objective: Develop a high throughput, multiplexed PCR-based platform for identification of a panel of avian respiratory viruses 1. Avian Influenza Virus (AIV) matrix 2. AIV H5 3. AIV H7 4. Newcastle Disease Virus (NDV) matrix 5. NDV fusion protein 6. Infectious Laryngotracheitis Virus (LTV) 7. Infectious Bronchitis Virus (IBV) ARVP identifies 7 virus types/subtypes of high priority to CFIA and USDA

11 Method Development Approach: Luminex xtag Technology Multiple DNA tests are combined and multiplexed simultaneously in a single PCR reaction Luminex xmap system uses lasers to read color-coded beads that attach to specific nucleic acid sequences

13 Luminex xtag Technology

15 Results Multiplex PCR of AVRP on Luminex xtag system Target Bead Region Sample Run 1 Run 2 Run 1 InflA-M InflA-H5 InflA-H7 APMV-F APMV-M IBV ILTV Run 2 Run 1 Run 2 Run 1 Run 2 Run 1 Run 2 Run 1 Run 2 Run 1 Run 2 InflA-M InflA-H InflA-H APMV-F APMV-M IBV ILTV Neg Background Background

16 Next steps Validate the efficacy of the assay using archived avian respiratory virus isolates Determine detection efficiency for co-infections with multiple targets in one sample Develop a diagnostic test and obtain CFIA approval for use at AHL and other reference labs

17 Impact Benefits of a multiplexed ARVP: Determines whether a respiratory infection is viral or bacterial Increases efficiency by providing data on multiple viral targets Reduces costs by minimizing repetition and increasing throughput Provides rapid results in outbreak situations Helps with surveillance and implementation of control measures Assists with clinical or regulatory decision-making

18 Acknowledgements Collaborators Dr. Davor Oykich, Virologist, AHL Dr. Gordon Kirby, ADRI, Anna Marom, Technician, AHL Sarah Hoyland, Technician, AHL Funding: OMAFRA

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