Point mutation revealed the resurgence of sub-genotype VII.2 Newcastle disease virus in Israel

Real-time reverse transcription PCR (rRT-PCR) is a widely used method for Newcastle disease virus (NDV) diagnosis and classification. The virus matrix (M) gene is usually used for general NDV identification and the fusion (F) gene for virulence differentiation. This method is rapid and sensitive, but the high mutation rate of the viral genome is a source of concern, as it may cause diagnostic failure. In late 2019, we encountered a problem classifying virulent NDV in samples from a few poultry flocks in which clinical signs were observed. Although the general NDV identification test was positive, the rRT-PCR virulence differentiation test gave negative results or higher cycle threshold values (Ct) than expected. Partial F gene sequencing for unclassified clinical NDV samples was used to investigate the cause of the rRT-PCR virulence differentiation test failure. The cause of the diagnostic failure was found to be a mismatch at the 3 ' end of the reverse primer and adjustments were made to the diagnostic assay. The partial sequencing also allowed us to differentiate the virus as virulent according to the amino acid sequence of the F gene cleavage site and to genetically classify the virus as sub-genotype VII.2 NDV. After realizing that this is a resurgence event of sub-genotype VII.2 virus in an already endemic area of sub-genotype VII.1, we traced the spread of the new virus for several months. In addition, whole-genome sequencing of two 2020 sub-genotype VII.2 isolates was performed. GenBank search yielded related sequences from Jordan (2018) and Pakistan (2015-2016).

Automated summary

Real-time reverse transcription PCR (rRT-PCR) is a method widely used for diagnosing and classifying Newcastle disease virus (NDV). This study found that the high mutation rate of the viral genome can cause diagnostic failure. A mismatch in the reverse primer was identified as the cause of the failure. Additionally, the virus was genetically classified and its spread was traced.