A novel diagnostic tool for the rapid pathotyping of type I avian paramyxovirus (APMV-1)

The World Organisation for Animal Health (WOAH) has listed the Newcastle disease (ND) as a disease affecting poultry worldwide. ND is caused by virulent avian paramyxovirus type I (APMV-1). The disease shows different clinical manifestations that may either affect the nerves, breathing and/or stomach, accompanied by high rate of death. In contrast, mild or subclinical forms are generally caused by lentogenic APMV-1 and are not subject to notification. The rapid differentiation of virulent and avirulent viruses is paramount to limit the spread of virulent APMV-1.

An in vivo pathogenicity test (intracerebral pathogenicity index, ICPI) is used as a starting ground to define a harmonized categorization of APMV-1 pathotype. As an alternative to ICPI, the WOAH has accepted sequencing of the cleavage site (CS) of the fusion protein gene precursor (F0) for ND case definition. This latter approach is largely preferred over ICPI determination, as it reduces both the use of live animals and costs, and shortens the time-to-results. However, developing countries, where APMV-1 is endemic in poultry and domestic birds, most often do not have sequencing facilities and are unable to rapidly identify the pathotype of the circulating NDVs.

In the framework of the EU-sponsored LIDISKI project, a new set of real-time RT-PCR (RT-qPCR) tests to identify virulent and avirulent viruses through pathotype-specific probes has been developed and validated. In particular, specific probes targeting the F gene CS and labelled with diverse dyes enable the discrimination of virulent and avirulent viruses. The size of the amplification product offers the chance to further confirm the pathotype assignment through Sanger sequencing and determination of the CS sequence. Validation was performed according to the WOAH recommendations and included the testing of a large panel of APMV-1 positive samples of different genotypes originating from Europe, Asia, the Middle East and Africa including those collected under the project from Nigeria.

Overall, the new RT-qPCRs showed its ability to detect and pathotype all tested APMV-1 genotypes and its suitability for routine use in clinical samples. To improve the ability of the entire veterinary diagnostician community to identify and type NDVs, the new real time RT-PCR protocol has been published open access in an international journal and is available at the following link.