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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Exotic & Emerging Avian Viral Diseases Research » Research » Research Project #438182

Research Project: Development and Validation of a Universal rReal-time RT-PCR Assay to Distinguish Between Virulent and Non-virulent APMV-1

Location: Exotic & Emerging Avian Viral Diseases Research

Project Number: 6040-32000-082-004-R
Project Type: Reimbursable Cooperative Agreement

Start Date: Mar 1, 2020
End Date: Sep 30, 2022

Newcastle disease (ND), caused by virulent strains of Avian paramyxovirus 1 (APMV-1), is a fatal infection in naïve chickens and is a threat to the poultry industry worldwide. Occurrence of virulent APMV-1 in commercial poultry results in trade restrictions. ND is considered a foreign animal disease for the U.S., however, outbreaks in poultry occasionally occur, including an ongoing outbreak in California. The disease has significant economic and social impact. Rapid and specific detection of virulent APMV-1 in suspected flocks is of critical importance for adequate and effective control and limiting costs and losses involved with outbreak containment. The current National Animal Health Laboratory Network (NAHLN) APMV-1 test that detects virulent strains was developed in response to the California 2002-2003 ND outbreak and has maximum sensitivity with genotype V viruses. However, APMV-1 are genetically diverse, constantly evolve and undergo simultaneous evolutionary changes. New emerging variants are circulating in Asia, the Middle East, Africa, Europe, and Central and South America and pose a constant threat to the U.S. poultry industry. The continuous evolution of these viruses presents diagnostic challenges and target-oriented assays might fail to detect new and emerging viral genetic variants. Testing APMV-1 isolates from different genotypes with the currently validated test has resulted in several published reports of either lower sensitivity or false negative results.

We propose to sequence previously uncharacterized avian paramyxovirus 1 (APMV-1) isolates. We will add their genetic sequences to a databank of all available sequences across all APMV-1 genotypes that we will create. These data will be utilized to perform a comprehensive in silico analyses and identify common sequence motifs for each APMV-1 sub/genotype. The aim is to develop new real-time RT-PCR test/s for detection of virulent APMV-1 (including the ones affecting pigeons and cormorants) with high sensitivity and specificity. This new assay will be evaluated at the bench for specificity and sensitivity with viruses representing all genotypes currently circulating in poultry and wild birds worldwide. We will utilize a wide spectrum of clinical samples to validate the new assay in five different laboratories. For high quality control of possible inhibition and extraction failure, we will design test/s utilizing exogenous internal positive control. An up-to-date sequence databank to be used by the National Veterinary Services Laboratory (NVSL) and the National Animal Health Laboratory Network (NAHLN) will also be created. To facilitate a rapid response in case of an outbreak of Newcastle disease (ND) in the U.S. caused by an emerging strain, a primers/probes bank will be developed. The new primers and probes will be aliquoted into working stocks and stored with sufficient volume to test at least 25,000 clinical samples. This relatively inexpensive diagnostic banking program can save valuable time in responding to an outbreak by providing ready-to-use reagents.