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Redesign and Validation of a Real-Time RT-PCR to Improve Surveillance for Avian Influenza Viruses of the H9 Subtype

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
OIE Reference Laboratory for Avian Influenza, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
Department of Animal Health, Ministry of Agriculture and Rural Development (MARD), Hanoi 115-19, Vietnam
Regional Laboratory for Animal Influenzas and Other Transboundary Animal Diseases, National Veterinary Research Institute (NVRI), Vom 930010, Nigeria
Laboratoire Central de l’Élevage (LABOCEL), Ministère de l’Agriculture et de l’Elevage, Niamey 485, Niger
Accra Veterinary Laboratory, Veterinary Services Directorate, Ministry of Food & Agriculture, Accra M161, Ghana
Laboratoire National de l’Élevage et de Recherches Vétérinaires (LNERV) de l’Institut Sénégalais de Recherches Agricoles (ISRA), Dakar-Hann 2057, Senegal
Laboratoire de Diagnostic Vétérinaire et de Sérosurveillance (LADISERO), Parakou 23, Benin
AI/ND National Reference Laboratory, Sciensano, 1050 Brussels, Belgium
Author to whom correspondence should be addressed.
Academic Editor: Feng Li
Viruses 2022, 14(6), 1263;
Received: 20 April 2022 / Revised: 1 June 2022 / Accepted: 4 June 2022 / Published: 10 June 2022
(This article belongs to the Special Issue Advances in Veterinary Virology)
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. View Full-Text
Keywords: avian influenza; H9Nx; molecular diagnosis; real-time RT-PCR; validation avian influenza; H9Nx; molecular diagnosis; real-time RT-PCR; validation
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MDPI and ACS Style

Panzarin, V.; Marciano, S.; Fortin, A.; Brian, I.; D’Amico, V.; Gobbo, F.; Bonfante, F.; Palumbo, E.; Sakoda, Y.; Le, K.T.; Chu, D.-H.; Shittu, I.; Meseko, C.; Haido, A.M.; Odoom, T.; Diouf, M.N.; Djegui, F.; Steensels, M.; Terregino, C.; Monne, I. Redesign and Validation of a Real-Time RT-PCR to Improve Surveillance for Avian Influenza Viruses of the H9 Subtype. Viruses 2022, 14, 1263.

AMA Style

Panzarin V, Marciano S, Fortin A, Brian I, D’Amico V, Gobbo F, Bonfante F, Palumbo E, Sakoda Y, Le KT, Chu D-H, Shittu I, Meseko C, Haido AM, Odoom T, Diouf MN, Djegui F, Steensels M, Terregino C, Monne I. Redesign and Validation of a Real-Time RT-PCR to Improve Surveillance for Avian Influenza Viruses of the H9 Subtype. Viruses. 2022; 14(6):1263.

Chicago/Turabian Style

Panzarin, Valentina, 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, and Isabella Monne. 2022. "Redesign and Validation of a Real-Time RT-PCR to Improve Surveillance for Avian Influenza Viruses of the H9 Subtype" Viruses 14, no. 6: 1263.

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