Introduction
Newcastle disease (ND) is a highly contagious disease with significant clinical impact and serious losses in poultry worldwide. The causative agent, Newcastle disease virus (NDV) or avian paramyxovirus type-1 (APMV-1) is a species within the genus Avian orthoavulavirus 1 , a member of the family Paramyxoviridae(Rima et al., 2019). The genome is composed of a negative-sense, single-stranded RNA of approximately 15 kb coding for at least seven proteins, in the 3’ to 5’ order nucleocapsid protein (NP), phosphoprotein (P), V protein (V), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN) and large RNA-dependent polymerase protein (L) (Steward et al., 1993).The HN protein recognizes and attaches to sialic acid receptors on the surface of permissive cells and mediates the fusion activity of the F protein at the cell membrane for release of the nucleocapsid complex into the cytoplasm (Lamb & Parks, 2007).
Based on its pathogenicity, NDV is categorized into four groups with increasing virulence from apathogenic, lentogenic, mesogenic and velogenic (Alexander, 2000). The molecular basis for NDV pathogenicity is associated with the amino acid sequence motif of the protease cleavage site of the fusion protein, and the abilities of specific cellular proteases to cleave this protein. Based on phylogenetic analysis, NDV can be distinguished in two distinct classes, 1 and 2, with one (1.I) and twenty one genotypes, respectively (2.I-2.XXI) (Dimitrov et al., 2019). Whereas the majority of class 1 viruses are avirulent and have their natural reservoir in aquatic wild birds (Kim et al., 2007), class 2 comprises virulent viruses that are responsible for the outbreaks in poultry and evolved over time(P. J. Miller et al., 2010). Phylogenetic evidence suggests that in the decades prior to the 1970s, genotypes 2.II, 2.III and 2.IV were predominant in North America, Asia and Europe, respectively. Only genotype 2.II comprises of lentogenic, mesogenic and velogenic pathotypes (Czegledi et al., 2002; Czegledi et al., 2003). and vaccines derived from lentogenic strains of this genotype (Seal et al., 1996). Genotype 2.VI viruses emerged in epizootics in the Middle East and Asia in the 1960s, while in the early 1970s genotype 2.V (most likely of South American origin) caused outbreaks in North America and in Europe (Herczeg et al., 2001; Wehmann et al., 2003). Later, genotype 2.VI viruses gave rise to strains adapted to pigeons, the so-called pigeon type paramyxovirus (PPMV-1) (D. J. Alexander et al., 1985). The new NDV nomenclature proposal (Dimitrov et al., 2019) placed subgroups 2.VIai and 2.VIaii, encompassing the early strains circulating in poultry (Chong et al., 2013), into the new genotypes 2.XX and 2.XXI whereas PPMV-1 is forming the β€œnew” genotype 2.VI with several sub-genotypes. Most recently, several sub-lineages of genotype 2.VII and 2.VIII emerged in the Far East and spread to further geographic areas (Herczeg et al., 1999). Genotype 2.VII is currently predominant among velogenic NDV and can be further subdivided i.e. with sub-lineages 2.VII.1.1 encompassing viruses that emerged in the 1990s in the Far East, Europe and Asia, and the Middle East; while 2.VII.1.2 viruses evolved in China, and circulated in South and East Africa, as well as in Israel and Pakistan (Dimitrov et al., 2019).
In Egypt, ND is endemic and despite extensive routine vaccination programs implemented in the commercial poultry farm sectors, new cases occur continuously, posing a threat to the national poultry industry. In Egypt, ND was identified for the first time in 1948 (Daubney & Mansy, 1948), and in the last few years NDV 2.II and 2.VII genotypes were reported from the country (Radwan et al., 2013). Also, co-infections with infectious bronchitis and avian influenza viruses were recorded in Egypt (Moharam et al., 2019; Naguib et al., 2017; Samy & Naguib, 2018). This has raised concerns regarding the efficacy of ND vaccination programs applied in the country. It has been hypothesized that, due to accumulated mutations as seen previously with highly pathogenic avian influenza virus (Grund et al., 2011), new NDV antigenic variants have arisen, that have an advantage to spread within a vaccinated population. In consequence, viruses that supersede previous virus populations should have a gain in fitness, here, mutations within antigenic relevant sites. To test this hypothesis, four isolates representing genotype 2-II (n=2), the old genotype 2.XXI (former 2.VIa; n=1) and the currently circulating genotype 2.VII.1.1 in Egypt (n=1) were genetically and antigenically analyzed, with special focus on the viral glycoproteins, that are facilitating receptor mediated virus uptake and are the main target for protective immune responses.