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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Exotic & Emerging Avian Viral Diseases Research » Research » Publications at this Location » Publication #425426
Research Project: Intervention Strategies to Predict, Prevent, and Control Emerging Strains of Virulent Newcastle Disease Viruses

Location: Exotic & Emerging Avian Viral Diseases Research

Title: Optimization and validation of a universal real-time RT-PCR assay to distinguish between virulent Newcastle disease viruses and viruses of low virulence

Author
item ALEXANDER MORRIS, ELLEN RUTH - Texas A&M Veterinary Medical Diagnostic Laboratory
item SCHROEDER, MEGAN - Texas A&M Veterinary Medical Diagnostic Laboratory
item ANDERSON, PHELUE - Texas A&M Veterinary Medical Diagnostic Laboratory
item SCHROEDER, LISA - Texas A&M Veterinary Medical Diagnostic Laboratory
item MONDAY, NICHOLAS - Texas A&M Veterinary Medical Diagnostic Laboratory
item GABRIEL, SENTIES-CUE - Texas A&M Veterinary Medical Diagnostic Laboratory
item FICKEN, MARTIN - Texas A&M Veterinary Medical Diagnostic Laboratory
item FERRO, PAMELA - Texas A&M Veterinary Medical Diagnostic Laboratory
item Suarez, David
item DIMITROV, KIRIL - Texas A&M Veterinary Medical Diagnostic Laboratory

Submitted to: Viruses
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/2/2025
Publication Date: 5/3/2025
Citation: Alexander Morris, E., Schroeder, M.E., Anderson, P.N., Schroeder, L.J., Monday, N., Gabriel, S., Ficken, M., Ferro, P.J., Suarez, D.L., Dimitrov, K.M. 2025. Optimization and validation of a universal real-time RT-PCR assay to distinguish between virulent Newcastle disease viruses and viruses of low virulence. Viruses. Viruses 2025, 17(5), 670. https://doi.org/10.3390/v17050670.
DOI: https://doi.org/10.3390/v17050670

Interpretive Summary: Newcastle disease is considered a foreign animal disease in the United States. We rely on having sensitive diagnostic tests to identify infected flocks and the USDA works with states to quickly eradicate the outbreak to prevent the virus from shedding. Real-time PCR is the most commonly used test in the United States. However the Newcastle disease virus (NDV) is extremely variable in sequence and for the PCR assay to work the test must be properly targeted to the virus. Although the PCR has worked well for most NDV isolates, we identified 3 groups of NDV where the sensitivity was lower than acceptable. Although these groups are not found in North America, we wanted to update the test so that it could accurately identify all NDV isolates. By comparing sequences of the different viruses we modified the original test to have better performance with all known NDV groups. These improvements will allow us to better detect any introductions of virus from anywhere in the world.

Technical Abstract: Newcastle disease is caused by virulent strains of Avian paramyxovirus 1 (APMV-1). Occurring worldwide, the disease has a significant social and economic impact. APMV-1, as an RNA virus, evolves rapidly with considerable genetic diversity and can be genetically divided into Class I and Class II groups with almost all virulent viruses being in Class II. The sequence differences adds complexity in maintaining high sensitivity and specificity of detection assays for molecular diagnostics. The current USDA’s fusion (F) gene rRT-PCR assay was designed for Class II AMPV-1 isolates with an emphasis on recent U.S. strains, and initial testing assessed for the detection of virulent Class II APMV-1 stains of all globally circulating genotypes. As previously described, some genotypes had lower detection sensitivity (genotype VII.1.1 and VII.2) or complete absence of detection (genotype XIV) were identified. Using bioinformatics approaches comparing a comprehensive complete F-gene sequence database, an additional forward primer and two probes were designed and evaluated. An exogenous internal positive control (XIPC) was added to the assay. The optimized multiplex assay successfully detected genotype XIV and had improved sensitivity for sub-genotypes VII.1.1 and VII.2, with maintained sensitivity for all remaining genotypes. No detection was observed in any of the tested near-neighbors or APMV-1 of low virulence. Using a large set of field and experimental clinical samples, both the specificity and sensitivity of the new assay were determined to be 100%, compared to the current assay (100% and 93%, respectively). The new F-gene rRT-PCR assay identifies all known virulent APMV-1 genotypes with the benefit of using an XIPC, which monitors extraction efficiency and rRT-PCR inhibitors.