Chumbe-Mendoza A.,AB# Izquierdo-Lara R.,AB# Calderón Mayo K.,A Fernández-Díaz M.,A Vakharia N. Vikram AC#. ALaboratorio de Biotecnología Molecular y Genómica, FARVET. BLaboratorio de Patología Aviar, Universidad Nacional Mayor de San Marcos, Facultad de Medicina Veterinaria, San Borja, Lima, Perú. C Institute of Marine & Envrironmental Technology, University of Maryland Baltimore County, EE.UU. #Estos autores contribuyeron igualmente para este trabajo.

Newcastle disease is one of the most important infectious diseases of the poultry industry, and is caused by the Newcastle disease virus (NDV). This virus is distributed worldwide and can cause serious losses in the bird industry economy due to recurrent outbreaks in vaccinated and unvaccinated chickens. Protection against NDV in chickens has been associated with the development of the humoral response. Although the hemagglutination (IH) inhibition assay and ELISA do not corroborate the presence of neutralizing antibodies (nAbs); they are used to measure protection and immune response against NDV.


In this study, we established a recovery system for a recombinant NDV (rLS1) from a cDNA clone, which is capable of accepting exogenous genes at the desired positions. A gene containing the enhanced green fluorescent protein (eGFP) was designed in the first position of the genome and we generated a recombinant NDV carrying the eGFP. The NDV-GFP reporter virus was used to develop a neutralization test based on the eGFP (eGFP-NT), in which the nAbs titers were expressed as the reciprocal titer of the highest dilution expressed by the eGFP.


The eGFP-NT gave conclusive results in 24 h without using any additional staining procedure. A total of 57 serum samples were analyzed by conventional neutralization (NT) and eGFP-NT. In addition, HI and a commercial ELISA kit were evaluated with the same set of samples. Although HI (R2 = 0.816) and ELISA (R2 = 0.791) showed substantial correlations with conventional NT, eGFP-NT showed a higher correlation (R2 = 0.994), indicating that eGFP-NT is a more accurate method to quantify nAbs.


In general, the neutralization test developed here is a simple, fast and reliable method for quantifying nAbs specific for NDV. It is suitable for vaccine evaluation and diagnostics.

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