Isolation, molecular detection and BHK-21 adaptation of Newcastle disease virus of field cases in layer farms of Bangladesh

In this study, field samples (n=45) such as trachea, brain and proventriculus were collected from 12 layer farms of different districts of Bangladesh for the detection and isolation of pathogenic Newcastle disease virus (NDV). Isolation of the virus from the clinical samples and their propagation was primarily carried out in chicken embryo. Among the forty five samples, 9 (20%) were found positive for NDV by hemagglutination (HA) and hemagglutination inhibition (HI) tests. The positive isolates were cultured in chicken embryo, and subsequently confirmed by reverse transcription-polymerase chain reaction (RT-PCR) targeting NDV specific ‘F’ gene that encodes fusion protein. A nested PCR was carried out with the RT-PCR product as the template, which targeted a smaller internal region of ‘F’ gene. The nested PCR also confirmed all the isolates as NDV. For further confirmation, NDV specific ‘F’ gene was also amplified from the cDNA generated from the RNA extracted from the virus inoculated chicken embryo by direct PCR. The isolates were inoculated in BHK-21 cell line and 2/3 blind passage were given without any gross change in the cell. Later, the virus was found adapted into BHK-21 cell line where they produced syncitia, rounding of cell and multinucleated giant cells as the cytopathic effects. Adaptation of the virus in BHK-21 cell line was confirmed by RT-PCR and nested-PCR successfully.