Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/31/2006
Publication Date: 11/14/2006
Citation: Kim, L.M., Afonso, C.L., Suarez, D.L. 2006. Effect of probe-site mismatches on detection of virulent Newcastle disease viruses using a fusion-gene real-time reverse transcription polymerase chain reaction test. Journal of Veterinary Diagnostic Investigation. 18:519-528.
Interpretive Summary: Newcastle disease virus is an important disease of poultry. The severe form of the virus can cause serious disease and often death in infected chickens, and it is considered a foreign animal disease in the United States. Although many efforts are used to prevent the virus from entering the U.S., outbreaks of virulent NDV occur every few years. One of the keys to controlling these outbreaks is to be able to rapidly diagnosis the infection. In recent years the availability of a rapid and sensitive diagnostic test, the real-time RT-PCR test, for NDV have aided control efforts. Although this test has proven to be a valuable test, previous work has shown that it does not detect all virulent NDV isolates. This study evaluated why an Italian Dove NDV isolate was not detected by looking at the sequence of the virus. It was determined that several changes in the sequence of the Dove isolate caused the test to miss detecting the virus. It was also determined that small changes in the existing test could be made so that the Italian dove isolate could be detected. Further work needs to be performed before these changes can be adopted.
Technical Abstract: Virulent forms of Newcastle disease virus (NDV) are a major concern for poultry producers around the world and the rapid diagnosis of an outbreak is crucial to any control program. A validated real-time reverse transcription-PCR test (Fusion test) directed at the fusion-cleavage site of NDV was developed to differentiate virulent Newcastle disease virus strains from those of low virulence, however one virulent isolate, Dove/Italy/2736/2000, escaped detection during the initial evaluation of the test. The objectives of this study were to determine how this isolate differed from other detectable isolates, to identify other isolates that may fail detection, and to characterize the effect of specific probe-site mutations on the Fusion test at a range of annealing temperatures. Using a virulent NDV isolate (Game fowl/US(CA)/2002) as a backbone which has 100% identity to the Fusion test probe, specific changes were made to the Fusion test probe-site to reflect the unique mismatches found in Dove/Italy/2736/2000 and other selected regions of the probe. Mutated clones with mismatches unique to Dove/Italy/2736/2000 at positions 6, 13 and 14 were not detected until annealing temperatures were lowered to 50°C. Those detected at 58°C contained 1-2 mismatches (position 1 and 6, 13 and 14, or 14 only) although increased cycle threshold values compared to the parent clone indicated decreased sensitivity. Data from this study predicts that the Fusion test may fail to detect some viruses among lineage 4b and potential solutions to identify this subset of viruses include lowering the annealing temperature or modifying the probe.