50 ISSN 0891-4168, Molecular Genetics, Microbiology and Virology, 2019, Vol. 34, No. 1, pp. 50–58. © Allerton Press, Inc., 2019. Developing a New Method for Pathotyping of Newcastle Disease Virus Based on Sialidase Protein V. Mayahi a , M. Esmaelizad b, *, and N. Harzandi a a Department of Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran b Central Laboratory Department, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Alborz, Iran *e-mail: m.esmaelizad@rvsri.ac.ir Received February 21, 2017; revised February 21, 2017; accepted June 29, 2018 Abstract—Newcastle disease virus (NDV) is considered as a deadly infection to majority of the avian species. One of the key factors responsible for virus pathogenesis is sialidase protein expressed by hemagglutinin- neuraminidase gene on the envelope of virus particles, which involves in virus attachment to cells. In this study we compared restriction map of hemagglutinin-neuraminidase gene encoding sialidase protein among Lentogenic strains and Velogenic isolates obtained in Iran, and a variety of virus genotypes (I–VII) retrieved from GenBank. A polymorphic region with 527 bp length was found in the mentioned gene. Reverse tran- scription-Polymerase chain reaction was done for Iranian Velogenic isolates and vaccine strains (B1, La Sota and V4). The Pattern of four restriction endonuclease enzymes (AluI, AvaIl, HaeIII and TaqI) was analyzed by Polymerase chain reaction-Restriction fragment length polymorphism technique. There were significant differences among Iranian Velogenic isolates and vaccine strains. The experimental results of the mentioned method were in concordance with in silico evaluations of restriction map of 146 strains including: class I and class II genotypes obtained from the National Center for Biotechnology Information (NCBI). Therefore, we suggest that this method might be useful for pathotyping and differentiation of class I and II Newcastle dis- ease virus. Keywords: Newcastle disease virus, pathogenesis, PCR-RFLP, molecular diagnosis DOI: 10.3103/S0891416819010063 INTRODUCTION Newcastle disease virus (NDV), the cause of fatal avian infection, has massive destructive impacts on poultry industry. Therefore, it is regarded as the factor of severe economic losses. This virus belongs to the genus Avulavirus as a member of the family Paramyxo- viridae. The single-stranded negative sense RNA of NDV with 15kb length is comprised of six genes encoding nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemag- glutinin-neuraminidase (HN), and RNA-dependent RNA polymerase (L). According to Avian paramyxo- virus serotype 1 (APMV-1) or NDV classification, NDVs are grouped into class I including nine geno- types and class II containing 18 genotypes (I–XVIII) [1]. Three main pathotypes are identified for APMV-1 depending on pathogenicity indices which are intracere- bral pathogenicity index (ICPI), intravenous pathoge- nicity index (IVPI), mean death time (MDT) including Lentogenic (avirulent), Mesogenic (intermediate), or Velogenic (virulent) [2]. Clinical signs and symptoms are widely different among NDV strains. NDVs can cause infections from mild symptoms of Lentogenic strains causing asymptomatic enteric infection or mild respiratory tract disease, to acute respiratory infection and neurological symptoms brought about by Mesogenic viruses of intermediate virulence. But the most fatal form of disease is caused by Velogenic strains dividing into two categories. [3]. Although F protein is considered as the main deter- minant of NDV infectivity, different studies have shown that HN protein is also important for pathoge- nicity. Hemagglutinin-neuraminidase protein is a transmembrane glycoprotein on the envelope of virus particles which plays a critical role in NDV pathogen- esis and also contributes to tissue tropism [4, 5]. The immunogenic trait of NDV is also derived from this protein. The process of NDV attachment to sialic acid receptors on the host cells, following neuraminidase (sialidase) activity, sialic acid cleavage, and releases of progeny virus particles from the surface of virions is mediated by HN protein. Hemagglutinin-neuramini- dase protein involves in facilitation of fusion by inter- action with F protein [5]. According to the high fatal- ity rate of NDV caused by Velogenic strains world- wide, it is of great importance to identify and distinguish avirulent and virulent strains. In this study we focus on molecular diagnosis of various vaccine EXPERIMENTAL WORKS