Among the destructive and vastly communicable viral infections of poultry the most devastating disease is Newcastle Disease (ND) caused by a virus belong to genus Avulavirus of paramyxoviridae family, avian paramyxovirus-1. The NDV causes huge economic losses to the Poultry Industry. The available vaccines failed to protect the birds from the disease; this project is designed to find reasons of the vaccine failures. Keeping in view the importance of outbreaks reported due to NDV and adverse effects on Poultry Industry a study was conducted to examine the function of the cleavage site of Fusion protein sequencing, in Newcastle Disease Virus virulency through Reverse Transcriptase Polymerase Chain Reaction via objective to find the genetic variations among the different isolates of Newcastle Disease field viruses in Lahore District.
One hundred suspected samples of NDV from dead and morbid birds were collected from different sources and areas of Lahore district. Prepared inoculums were inoculated in the 9-11 days old embryonated hen eggs for virus isolation. The allontoic amniotic fluid (AAF) was harvested and tested for HA activity. Further confirmation of NDV was done by using Reference NDV antiserum (HI). Out of 100 samples, 63 showed Hemagglutination activity with washed chicken RBCs and only 16 samples were repressed with specific known NDV antiserum. The remaining samples showed inhibition with known H5, H7 and H9 specific antiserum (12, 13, and 22) respectively. The isolates that were found to be positive through Hemagglutination Inhibition Test (HI) were further tested for Intra Cerebral Pathogencity Index (ICPI). ICPI was performed to characterize the isolates into Lentogenic, Mesogenic and Velogenic forms. The ICPI values obtained after pathogencity test of 16 isolates showed that only 6 isolates have the pathogenicity index above 1.5, and the remaining isolates below 1.5 and above 1, the average higher ICPI value of 16 virus isolates was 1.78.
On the basis of Intracerebral Pathogencity index (ICPI) results only 5 samples were selected for RNA extraction and PCR amplification. The RNA extraction was performed by using kit method (High Pure RNA Isolation kit by Roche-Germany) as recommended by the manufacturer. The gene representing F protein was amplified through Reverse transcriptase polymerase chain reaction (RT-PCR).

Nucleotide sequencing of complete (1580 bp) F gene of 1 NDV isolate was performed. The sequencing results of 1580 bp were compiled and sequence alignment of the NDV isolates, based on a variable portion covering the F-gene site, was done by using, software, ClustalW. The Neighbor-joining phylogenetic Tree was constructed with bootstrap value 1000 using software, MEGA 4.1. The phylogenetic result showed that our isolate has been distinct from Pakistani isolate and has 96% similarity with SPVC/Karachi/33/2007 (velogenic) available in GenBank.