SNP analysis used to select conserved regions for an improved real-time RT-PCR test specific to Newcastle disease virus

Authors: FERREIRA, HELENA - Orise Fellow; Suarez, David

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/12/2019
Publication Date: 8/12/2019
Citation: Ferreira, H., Suarez, D.L. 2019. SNP analysis used to select conserved regions for an improved real-time RT-PCR test specific to Newcastle disease virus. Avian Diseases. 63:625-633. https://doi.org/10.1637/aviandiseases-D-19-00071.
DOI: https://doi.org/10.1637/aviandiseases-D-19-00071

Interpretive Summary: Newcastle disease viruses (NDV) continue as an ongoing threat to the poultry industry and are reportable to the World Organization for Animal Health. The rapid detection of NDV, typically using a molecular amplification method called real-time reverse transcription PCR (RRT-PCR), is critical for early detection and fast control response. Many countries have adopted a strategy of using RRT-PCR screening tests that try to identify any NDV virus targeting their most conserved genes. The first widely used RRT-PCR screening test, targeting the matrix gene, was developed in 2002 during an outbreak in the USA. Although the matrix RRT-PCR test was designed to identify all NDV viruses, it has subsequently been shown to have lower sensitivity for some NDV strains. This study aimed to design new RRT-PCR tests for NDV diagnosis utilizing the vastly larger sequence database available currently to identify conserved regions along the NDV genome. Three new RRT-PCR tests were developed and optimized and these tests were compared with two other commonly used RRT-PCR tests, including the matrix test. The matrix test was compared with the new tests on a panel of 46 NDV isolates from different countries and the sensitivity of these tests was also compared. The present study suggested the established tests are still useful tools to detect all viruses, with a variable sensitivity. The new RRT-PCR tests can be used as alternative detection tools that have high sensitivity and specificity. The availability of alternative bench validated tests for NDV provides flexibility if a problematic NDV isolate is circulating in the region.

Technical Abstract: A bioinformatics approach using single-nucleotide polymorphism (SNP) analysis was performed to improve the current real-time reverse transcription–PCR (RRT-PCR) tests for the rapid detection of Newcastle disease virus (NDV). In total, 422 NDV complete genomes were analyzed using the Virus Pathogen Resource to compare the conservation of the primer and probe sequences and to select regions to develop new RRT-PCR tests. The sensitivity and specificity of the three new RRT-PCR tests targeting the nucleoprotein (NP) and polymerase (L) genes were optimized and were compared with established tests for NDV detection. The SNP analysis was also used to identify the number of mismatches between selected primers/probes and the NDV complete genome sequences. The SNP analysis, averaged over the entire primer or probe, showed the primer/probe sequences of three new tests were more conserved than the primer/probe sequences of the commonly used test targeting the matrix (M) gene. The M RRT-PCR test was compared with the new tests on a panel of 46 viruses, comprising 31 NDV isolates. Limit of detection (LOD) varied from 1.3 to 3.7 log 50% egg-infective doses using five isolates from different genotypes by all tests. The two RRTPCR tests targeting the L and M genes detected three out of five isolates with the lowest LOD. The NP and M RRT-PCR tests had the lowest and highest rates of genetic variants, respectively, among all probes. Because currently used tests are likely to miss some isolates, the availability of validated alternative tests provides alternatives for detection of viral variants that can be rapidly deployed to diagnostic laboratories.