SUPPLEMENTARY MATERIAL AVAILABLE ONLINE Indian Journal of Animal Sciences 93 (6): 549–555, June 2023/Article https://doi.org/10.56093/ijans.v93i6.132450 Genomic and biological characterization of Pigeon Paramyxovirus Type-1 isolated from Indian pigeons: First report on the six nucleotide insertion in the non-coding region of nucleoprotein gene V S AKHILA 1 , J JOHN KIRUBAHARAN 1* , RANJANI RAJASEKARAN 2 , P SHILPA 3 , M VIDHYA 1 and S RAJALAKSHMI 1 Madras Veterinary College, Chennai, Tamil Nadu 600 007 India Received: 19 January 2023; Accepted: 19 April 2023 ABSTRACT Genotypic analysis of Pigeon Paramyxovirus Type-1 (PPMV-1) virus, in corroboration with pathotyping, underpins the knowledge on the occurrence of PPMV-1 mediated Newcastle disease in poultry. In the present study, two PPMV-1 isolates, namely D167 and D168, obtained from feral pigeons of India in the year 2014 were analysed for their genotypic and pathotypic characteristics. Phylogenetic analysis of Fusion (F) gene grouped D167 and D168 under sub-genotypes VI.2.2.2. and VI.2.1.1.2.2. respectively. The separate positioning of these isolates within genotype VI is corroborated with the variation in restriction enzyme sites analysed through virtual restriction mapping. Analysis of FPCS region showed that both D167 and D168 possessed 112 RRQKRF 117 amino-acid pattern. Intra-cerebral pathogenicity index (ICPI) of D167 and D168 was 1.57 and 1.3, and mean death time (MDT) was 48 and 70 hrs respectively, designating D168 as a mesogenic pathotype, and D167 as a velogenic pathotype. Histopathological analysis of proventriculus, intestine, lungs and spleen of chickens infected with D167 and D168 isolates through intramuscular (I/M), subcutaneous (S/C) and oculo-nasal/oral (O/N) routes of inoculation showed similar severity of tissue damage. This study shows that PPMV-1 isolates belonging to two different sub-genotypes without a common ancestor, can occur together in a particular geographical region. It also shows that PPMV-1 gains virulence in chickens, which needs to be addressed in order to avoid emergence of PPMV-1 as new velogenic variant causing Newcastle disease in chickens. Keywords: Avian Orthoavulavirus 1, FPCS analysis, Genotyping, Newcastle disease, Pathotype, Pigeon Paramyxovirus Present address: 1 Department of Veterinary Microbiology, Madras Veterinary College, Chennai, Tamil Nadu. 2 Department of Veterinary Microbiology, Veterinary College and Research Institute, TANUVAS, Theni, Tamil Nadu. 3 Veterinary Polyclinic, Kodugallur, Thrissur, Kerala. * Corresponding author email: jjohnk@gmail.com Pigeon Paramyxovirus–1 (PPMV-1) is a variant of Avian Orthoavulavirus–1 (AOaV-1) that causes Newcastle Disease (ND) in pigeons and is categorized under Genotype VI of AOaV-1 of Orthoavulavirus genus of Paramyxoviridae family. It has been speculated that multiple events of interspecies transmission of Genotype VI viruses of AOaV-1 between pigeons and chicken might have led to the origination of PPMV-1 in North East Africa (Ujvari et al. 2003) and in India (Mangat et al. 1988). Spill-over events of PPMV-1 from pigeons to poultry necessitated the need to study these viruses to understand their evolution dynamics. PPMV-1 is an enveloped virus encompassing a single stranded, non-segmented, negative sense RNA genome. The complete genome of PPMV-1 consists of 15,192 nucleotides coding for six structural proteins (Millar and Emmerson 1988, Lamb and Parks 2007) namely nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin-neuraminidase (HN) and large polymerase (L). The 5’ non-coding region of NP gene of PPMV-1 comprises an additional six nucleotides (6-nt), in contrast to the classical AOaV-1 genome (Phillips et al. 1998, Huang et al. 2004). The presence of additional 6-nt was observed between 1647 and 1648 nucleotide position of classical AOaV-1 genome (Huang et al. 2004, Shan et al. 2021). The amino acid sequence of Fusion protein cleavage site (FPCS) of PPMV-1 strains isolated so far consists either of 112 GRQKRF 117 or 112 RRKKRF 117 or 112 RRQKRF 117 , characteristic of velogenic strains (Collins et al. 1994). However, their pathogenicity in chickens range from moderate to no virulence (Dortmans et al. 2010). In the recent past, PPMV-1 has been reported to gain virulence over passages in chicken or embryonated chicken egg (Kommers et al. 2003), which warrants the study of genotypic and pathotypic characteristics of PPMV- 1 isolates. This will aid in understanding the evolution patterns of PPMV-1 and the possibilities of occurrence of PPMV-1 mediated ND in chickens. Therefore, in the present study, two PPMV-1 isolates, namely D167 and D168 isolated from pigeons in South India were subjected 9