First report of the emergence of novel sub-genotype XIII.2.3 of Newcastle disease virus in chickens from selected regions of Bangladesh

Newcastle disease (ND) is one of the most economically devastating infectious diseases impacting the poultry industry in Bangladesh. This study aimed to characterize the pathotype, genotype, evolutionary divergence, and mutations of circulating virulent Newcastle disease virus (NDV) in chickens from the Gazipur, Tangail, and Mymensingh districts of Bangladesh between October 2023 and December 2024. ND-suspected samples, including lung, trachea, and caecal tonsil tissues, were collected, processed, and inoculated into 10-12-day-old embryonated chicken eggs (ECEs) via the allantoic cavity. Allantoic fluids were harvested after 24 h of incubation, and virulent NDV was identified through RT-PCR targeting the fusion (F) gene using specific primers. Pathogenicity was assessed using the mean death time (MDT), intracerebral pathogenicity index (ICPI), and intravenous pathogenicity index (IVPI). The pathotype and genotype were confirmed by complete sequencing of the F gene and phylogenetic analysis. Further evolutionary divergence and mutations were analyzed using MEGA-11 software. RT-PCR yielded specific amplification of a 254-bp product indicative of virulent NDV. Pathogenicity indices-MDT (<60 h), ICPI (>1.5), and IVPI (>1.70)-confirmed a velogenic strain. Complete F gene sequencing revealed an F-protein cleavage site motif of "RRQKRF," while phylogenetic analysis classified the isolates as belonging to sub-genotype XIII.2.3 under genotype XIII. Evolutionary divergence (0.00-0.06) and mutations at neutralizing epitopes 1 and 2 (at the 74th and 170th amino acids, respectively) suggested moderate genetic diversity. This study represents the first report in Bangladesh identifying the emergence of the novel sub-genotype XIII.2.3 of genotype XIII NDV associated with chicken mortality in selected regions.