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Background: In reverse genetics studies, minigenomes of negative sense RNA viruses are used to model the process of virus replication and evaluate the virus rescue system in the course of construction of recombinant virus. In the current study, a minigenome (MG) rescue system for lentogenic D58 vaccine strain of Avian orthoavulavirus 1 (AOAV-1) (Newcastle disease virus) was developed to establish a reverse genetic system for this strain. Methods: The minigenome was constructed by assembling the trailer and leader regions of AOAV-1 D58 strain flanking the reporter gene, Aequorea coerulescens green fluorescence protein (AcGFP) in a modified transcription vector. Construction of this Minigenome was done using a simple restriction free technique-Splicing by Overlap Extension PCR. The constructed minigenome was evaluated for the reporter gene expression by transfection in T7 RNA polymerase expressing BSR/T7 cell line. Further, the replication and packaging ability of the constructed minigenome were also analyzed. Result: The reporter gene expression was confirmed in BSR/T7 cells which simulates the virus replication process and verifies the virus rescue system. In addition, this proves the applicability of restriction free cloning by Splicing by Overlap Extension (SOE) PCR for precise construction of the minigenome. The developed minigenome system can be used as a potential tool in the reverse genetic rescue of AOAV-1 D58 strain for the development of marker vaccine. [ABSTRACT FROM AUTHOR]

Knowledge on the influence of pro-inflammatory cytokine and apoptotic gene mRNA levels in the pathogenesis of Indian field isolates of Newcastle disease virus (NDV) is little. In this study, cytokine mRNA levels were elucidated in spleen of chickens (in-vivo) and chicken embryo fibroblast cells (in-vitro) infected with lentogenic D58 strain and viscerotropic velogenic D165 isolate until five days post infection (dpi). In spleen of chickens infected with D165, maximum upregulation of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), chemokine (IL-8) and apoptotic gene (Caspase-8) at 3dpi correlated with the onset of severe clinical signs and necrotic histopathological lesions in spleen, proventriculus, intestine and caecal tonsil of chickens. Similarly, in CEF cells infected with D165, upregulation of pro-inflammatory cytokine and apoptotic gene mRNA levels correlated with the appearance of CPE. In spleen of chickens and CEF cells infected with D58, there was comparatively minimal upregulation of pro-inflammatory cytokine and apoptotic gene mRNA levels which did not cause histopathological changes in tissues and CPE formation in CEF cells. In both in-vivo and in-vitro systems, upregulation of anti-inflammatory cytokine IL-10 showed inhibitory effects on the mRNA levels of pro-inflammatory cytokines. Thus, this study reports variation in the cytokine mRNA levels elucidated in response to two different pathotypes isolated from India and associates the same with the clinical signs and pathological lesions produced during the course of ND. [ABSTRACT FROM AUTHOR]