Redesign and Validation of a Real-Time RT-PCR to Improve Surveillance for Avian Influenza Viruses of the H9 Subtype
Valentina Panzarin,
Sabrina Marciano,
Andrea Fortin,
Irene Brian,
Valeria D’Amico,
Federica Gobbo,
Francesco Bonfante,
Elisa Palumbo,
Yoshihiro Sakoda,
Kien Trung Le,
Duc-Huy Chu,
Ismaila Shittu,
Clement Meseko,
Abdoul Malick Haido,
Theophilus Odoom,
Mame Nahé Diouf,
Fidélia Djegui,
Mieke Steensels,
Calogero Terregino,
Isabella Monne
Affiliations
Valentina Panzarin
EU/OIE/National Reference Laboratory for Avian Influenza and Newcastle Disease, FAO Reference Centre for Animal Influenza and Newcastle Disease, Division of Comparative Biomedical Sciences, Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), 35020 Legnaro, Italy
Sabrina Marciano
EU/OIE/National Reference Laboratory for Avian Influenza and Newcastle Disease, FAO Reference Centre for Animal Influenza and Newcastle Disease, Division of Comparative Biomedical Sciences, Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), 35020 Legnaro, Italy
Andrea Fortin
EU/OIE/National Reference Laboratory for Avian Influenza and Newcastle Disease, FAO Reference Centre for Animal Influenza and Newcastle Disease, Division of Comparative Biomedical Sciences, Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), 35020 Legnaro, Italy
Irene Brian
EU/OIE/National Reference Laboratory for Avian Influenza and Newcastle Disease, FAO Reference Centre for Animal Influenza and Newcastle Disease, Division of Comparative Biomedical Sciences, Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), 35020 Legnaro, Italy
Valeria D’Amico
EU/OIE/National Reference Laboratory for Avian Influenza and Newcastle Disease, FAO Reference Centre for Animal Influenza and Newcastle Disease, Division of Comparative Biomedical Sciences, Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), 35020 Legnaro, Italy
Federica Gobbo
EU/OIE/National Reference Laboratory for Avian Influenza and Newcastle Disease, FAO Reference Centre for Animal Influenza and Newcastle Disease, Division of Comparative Biomedical Sciences, Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), 35020 Legnaro, Italy
Francesco Bonfante
EU/OIE/National Reference Laboratory for Avian Influenza and Newcastle Disease, FAO Reference Centre for Animal Influenza and Newcastle Disease, Division of Comparative Biomedical Sciences, Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), 35020 Legnaro, Italy
Elisa Palumbo
EU/OIE/National Reference Laboratory for Avian Influenza and Newcastle Disease, FAO Reference Centre for Animal Influenza and Newcastle Disease, Division of Comparative Biomedical Sciences, Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), 35020 Legnaro, Italy
Yoshihiro Sakoda
OIE Reference Laboratory for Avian Influenza, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
Kien Trung Le
OIE Reference Laboratory for Avian Influenza, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
Duc-Huy Chu
Department of Animal Health, Ministry of Agriculture and Rural Development (MARD), Hanoi 115-19, Vietnam
Ismaila Shittu
Regional Laboratory for Animal Influenzas and Other Transboundary Animal Diseases, National Veterinary Research Institute (NVRI), Vom 930010, Nigeria
Clement Meseko
Regional Laboratory for Animal Influenzas and Other Transboundary Animal Diseases, National Veterinary Research Institute (NVRI), Vom 930010, Nigeria
Abdoul Malick Haido
Laboratoire Central de l’Élevage (LABOCEL), Ministère de l’Agriculture et de l’Elevage, Niamey 485, Niger
Laboratoire National de l’Élevage et de Recherches Vétérinaires (LNERV) de l’Institut Sénégalais de Recherches Agricoles (ISRA), Dakar-Hann 2057, Senegal
Fidélia Djegui
Laboratoire de Diagnostic Vétérinaire et de Sérosurveillance (LADISERO), Parakou 23, Benin
Mieke Steensels
AI/ND National Reference Laboratory, Sciensano, 1050 Brussels, Belgium
Calogero Terregino
EU/OIE/National Reference Laboratory for Avian Influenza and Newcastle Disease, FAO Reference Centre for Animal Influenza and Newcastle Disease, Division of Comparative Biomedical Sciences, Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), 35020 Legnaro, Italy
Isabella Monne
EU/OIE/National Reference Laboratory for Avian Influenza and Newcastle Disease, FAO Reference Centre for Animal Influenza and Newcastle Disease, Division of Comparative Biomedical Sciences, Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), 35020 Legnaro, Italy
Avian influenza viruses of the H9 subtype cause significant losses to poultry production in endemic regions of Asia, Africa and the Middle East and pose a risk to human health. The availability of reliable and updated diagnostic tools for H9 surveillance is thus paramount to ensure the prompt identification of this subtype. The genetic variability of H9 represents a challenge for molecular-based diagnostic methods and was the cause for suboptimal detection and false negatives during routine diagnostic monitoring. Starting from a dataset of sequences related to viruses of different origins and clades (Y439, Y280, G1), a bioinformatics workflow was optimized to extract relevant sequence data preparatory for oligonucleotides design. Analytical and diagnostic performances were assessed according to the OIE standards. To facilitate assay deployment, amplification conditions were optimized with different nucleic extraction systems and amplification kits. Performance of the new real-time RT-PCR was also evaluated in comparison to existing H9-detection methods, highlighting a significant improvement of sensitivity and inclusivity, in particular for G1 viruses. Data obtained suggest that the new assay has the potential to be employed under different settings and geographic areas for a sensitive detection of H9 viruses.
