INTRODUCTION

Viral diseases continue to pose a serious threat to animal health and poultry production worldwide, causing recurrent outbreaks with substantial economic losses and adverse impacts on food security [1–5]. Most of these outbreaks are caused by RNA viruses, which are characterized by high mutation rates and rapid evolution, facilitating viral adaptation, transmission, and persistence, particularly in backyard poultry production systems [6–9]. Among these pathogens, Newcastle disease virus (NDV) remains one of the most economically important avian viruses because of its high transmissibility, rapid spread, elevated mortality, impaired growth performance, and reduced egg production [10]. Previously known as avian paramyxovirus-1, NDV is currently classified by the International Committee on Taxonomy of Viruses as Avian orthoavulavirus 1, belonging to the genus Orthoavulavirus, subfamily Paramyxovirinae, and family Paramyxoviridae [11]. NDV possesses an enveloped, single-stranded, negative-sense, non-segmented RNA genome of approximately 15 kilobases encoding eight viral proteins, including the two major surface glycoproteins, fusion (F) and hemagglutinin-neuraminidase (HN) [12, 13]. The hemagglutinin-neuraminidase glycoprotein mediates viral attachment to host cells, whereas the F protein facilitates membrane fusion, viral entry, and hemolytic activity [13–16].

The clinical severity of NDV infection varies considerably depending on viral strain, concurrent infections, immune status, bird age, environmental conditions, and host species, with mortality rates reaching 100% during outbreaks caused by highly virulent strains [17, 18]. Based on pathogenicity and clinical manifestations, NDV strains are classified into four pathotypes: lentogenic strains, which generally cause subclinical or mild respiratory disease and are widely used as vaccine strains; mesogenic strains, which produce moderate respiratory disease, decreased egg production, and occasional neurologic signs; velogenic strains, which cause severe systemic disease associated with high mortality; and asymptomatic enteric strains, which produce inapparent intestinal infections [19, 20]. In Egypt, NDV control primarily depends on vaccination programs and strict biosecurity measures using live, inactivated, and genetically modified vaccines, predominantly derived from genotypes I and II. However, the continuous genetic evolution of NDV compromises vaccine efficacy and remains a major challenge for disease control [19, 20].

NDV virulence is primarily determined by the amino acid composition of the F protein cleavage site, where proteolytic activation of the precursor F protein is essential for viral infectivity [21]. Velogenic strains possess multiple basic amino acids at the cleavage site, allowing cleavage by ubiquitous intracellular proteases and facilitating systemic dissemination. In contrast, lentogenic strains contain monobasic cleavage motifs that restrict viral replication largely to the respiratory and intestinal mucosa [21]. Based on F gene sequencing and phylogenetic analysis, NDV isolates are classified into two major classes despite belonging to a single serotype. Class I comprises predominantly nonvirulent viruses circulating in wild aquatic birds, whereas class II includes most virulent viruses infecting domestic poultry and is currently divided into twenty-one recognized genotypes (I–XXI) [10, 20]. Since the late 1980s, genotype VII has been the predominant lineage responsible for the fourth global NDV pandemic and remains the principal genotype associated with outbreaks in Egypt despite intensive vaccination programs [22–24]. Nevertheless, genotypes II and VI continue to circulate in Egypt and other parts of North Africa, highlighting the dynamic molecular epidemiology of NDV in the region [25].

NDV has a remarkably broad host range and infects more than 250 avian species, including ostriches (Struthio camelus) [26]. In Egypt, ostrich farming has expanded due to its commercial value; however, the industry continues to incur substantial production losses from low hatchability, embryonic mortality exceeding 30%, poor chick survival, and infectious diseases [27, 28]. NDV was first reported in Egyptian ostrich farms in 2010, where outbreaks were associated with high mortality and neurologic manifestations in susceptible birds. Infection in ostriches represents an important veterinary and economic concern because it reduces productivity, facilitates viral maintenance within mixed-species production systems, and may contribute to disease transmission among ostriches, commercial poultry, and wild birds, thereby posing additional biosecurity challenges [28–30].

Although NDV has been extensively investigated in chickens and other domestic poultry, information regarding its molecular epidemiology, biological characteristics, and pathological manifestations in ostriches remains scarce. Published studies have largely focused on outbreak descriptions or on the detection of virulent NDV strains, whereas comprehensive investigations that integrate virus isolation, biological pathotyping, molecular detection, phylogenetic characterization, and histopathological evaluation of naturally infected ostriches are lacking. Furthermore, the circulating genotypes, molecular pathotypes, and tissue lesions associated with naturally occurring lentogenic genotype II NDV in Egyptian ostriches remain poorly understood. This knowledge gap limits understanding of host-specific disease expression, the epidemiological role of ostriches in NDV maintenance and transmission, and the implications of vaccine-related genotype II viruses for surveillance and control strategies in Egypt.

Therefore, this study aimed to comprehensively characterize NDV circulating in naturally infected young ostrich flocks in Egypt through virus isolation, biological pathotyping, molecular detection by real-time reverse-transcription polymerase chain reaction (rRT-PCR), partial F gene sequencing, phylogenetic analysis, and histopathological examination. The study further sought to determine the prevalence, genotype, pathotype, and phylogenetic relationships of circulating NDV isolates, evaluate the associated pathological alterations in major organs, and investigate the presence of coinfections with other economically important avian viruses. To the best of our knowledge, this study represents the first comprehensive molecular, biological, phylogenetic, and histopathological characterization of naturally circulating lentogenic genotype II NDV in young Egyptian ostriches, providing valuable baseline information for molecular surveillance, host-specific vaccination strategies, and improved biosecurity programs for the ostrich industry in Egypt.