Genetic analysis of Newcastle disease virus from Punjab, Pakistan Muhammad Zubair Shabbir • Muhammad Abbas • Tahir Yaqub • Nadia Mukhtar • Atta Subhani • Hasham Habib • Muhammad Umar Sohail • Muhammad Munir Received: 10 August 2012 / Accepted: 1 December 2012 Ó Springer Science+Business Media New York 2012 Abstract The strains of Newcastle disease virus (NDV) were isolated from five suspected outbreaks of ND in broiler (n = 3) and layers (n = 2) poultry farms. The egg- isolated viruses were subjected to biological and genetic characterization. Based on the biological characterization, isolates showed haemagglutination titer Clog 2 7 , mean death time \ 55 h, intracerebral pathogenecity index B1.8, and egg lethal dose 50 from 10 -7.15 to 10 -9.31 /1 ml. Genetic characterization of the fusion (F) gene revealed that the isolates clustered with NDV strains of genotype VII (VIIf) within class II, which remained predominant genotype in the domestic poultry of Asia. The deduced amino acid sequence of the isolates confirmed virulent motif 112 RRQKRF 117 at the F protein cleavage site. A bioinformatics and pairwise comparison approach was applied to estimate the synonymous and non-synonymous substitution rates (dN/dS) and selective evolutionary pres- sure for the F protein. The dN/dS calculated for genotype VII indicate purifying selection, which resulted in a low evolution rate in F gene. The F protein shows a strong negative pressure throughout the length of the protein and no recombination event was determined. Collectively, the results suggest that very similar virulent strains of NDV are involved during current wave of disease outbreak throughout the country. From these results, in conjunction with our recent reports of NDV from Pakistan, it is possible to conclude that emergence of novel group may require revisiting the diagnostics and vaccine control strategies. Keywords Newcastle disease Á Fusion protein Á Phylogenetic analysis Á Evolutionary analysis Á Pakistan Introduction Newcastle disease (ND) is a highly contagious disease of the birds caused by avian paramyxovirus type 1 (APMV-1) [1]. All avian species are susceptible; however, disease conse- quences are significantly higher in domestic poultry due to susceptibility to virulent strains [2]. Therefore, it is known to have serious economic losses to the poultry industry around the globe than any other viral diseases of poultry [1]. The NDV genome is approximately 15 kb long that encodes six proteins namely nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin–neuraminidase (HN), and large RNA- dependent polymerase protein (L) in the order 3 0 -NP-P-M- F-HN-L-5 0 [1]. Within a single serotype, the NDV exist in two distinct classes, class I and class II [3]. Class II strains Muhammad Zubair Shabbir and Muhammad Abbas equally contributed as first authors. M. Z. Shabbir University Diagnostic Laboratory, University of Veterinary and Animal Sciences, Lahore 54600, Pakistan M. Abbas Veterinary Research Institute, Lahore, Pakistan T. Yaqub Á N. Mukhtar Á H. Habib WTO-Quality Operations Laboratory, University of Veterinary and Animal Sciences, Lahore, Pakistan A. Subhani Veterinary Officer Health, Livestock and Dairy Development Department, Faisalabad, Punjab, Pakistan M. U. Sohail Islamia University, Bahawalpur, Pakistan M. Munir (&) Swedish University of Agricultural Sciences, Uppsala, Sweden e-mail: drmunir.muhammad@gmail.com 123 Virus Genes DOI 10.1007/s11262-012-0862-2