Melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2) are RNA-sensing PRRs expressed in the cytoplasm that play a key role in the activation of the viral sensing pathway [23,24]. hemagglutinin-neuraminidase (HN) glycoproteins compared to NDV LaSota. These structural differences were accompanied by increased hemagglutinating and neuraminidase activities of rNDV-H5. During in vitro rNDV-H5 infection, increased mRNA expression of TLR3, TLR7, MDA5, and LGP2 was observed, suggesting that the recombinant virus is recognized differently by sensors of innate immunity when compared with the parental NDV LaSota. Given the growing interest in using NDV as a vector against human and animal diseases, these data highlight the importance of thoroughly understanding the recombinant vaccines structural organization, functional characteristics, and elicited immune responses. family, NDV is known to enter its target cell through direct fusion with the CMPDA cell membrane, and it has been suggested to use a caveolae-dependent endocytic pathway as an alternative route for viral entry [16,17]. A previous study examining a recombinant NDV expressing the glycoprotein GP of the Ebola virus showed that it used GP-dependent macropinocytosis as a major cell entry pathway, indicating that the foreign GP can function as an entry protein [18]. The AIV entry process begins with the binding of the hemagglutinin (HA) to sialic acids at the cell surface and the internalization of the viral particle by endocytosis. The low pH within the endosome triggers conformational changes in the HA, exposing the fusion peptide and inducing the fusion between CMPDA the virus and the endosomal membrane [19]. The ability of rNDV-H5 to use an H5-dependent entry pathway under certain conditions, such as the presence of ND maternal antibodies, has been suggested by a previous study [20]. The use of this alternative entry pathway could, therefore, affect vaccine-induced immune responses. Because the latter may differ from the immune responses induced by the parental NDV LaSota, their characterization would improve the understanding of protection outcomes previously FAZF observed with rNDV-H5 immunization. In this study, the analyses focused on innate responses known to be involved in the regulation and orientation of subsequent adaptive responses [21]. Pathogen recognition by innate immune system is mediated through the sensing by pattern recognition receptors (PRRs). The activation of these receptors generates signals that trigger intracellular cascades resulting in the production of key soluble mediators that influence the polarization of adaptive immune responses [22]. Among PRRs, toll-like receptors (TLRs) -3 (TLR3) and -7 (TLR7) are important virus sensors capable of recognizing nucleic acids in intracellular compartments such as endosomes. Melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2) are RNA-sensing PRRs CMPDA expressed in the cytoplasm that play a key role in the activation of the viral sensing pathway [23,24]. The recognition of nucleic acids derived from pathogens during an infection ultimately leads to the production of type-I interferons (IFNs) that mediate the antiviral response [25] and cytokines that influence the polarization of adaptive immune responses [26,27]. To determine if the recombinant NDV-H5 retains the structural and functional characteristics of the parental NDV LaSota strain, the present study compared the structural organization and enzymatic activity of surface glycoproteins, and the recognition of both viruses by innate immune sensors. 2. Materials and Methods 2.1. Chickens SPF White Leghorn chickens were hatched from embryonated eggs purchased from Lohmann Valo (Cuxhaven, Germany). After hatching, the chickens were housed in biosecurity level 3 isolators. Feed and water were provided ad libitum throughout the experimental period. 2.2. Vaccines and Viruses CMPDA The rNDV-H5 vaccine expressing a modified H5 ectodomain of human HPAI H5N1 clade 1 A/Vietnam/1203/04, and the NDV LaSota were provided by Lohmann Animal Health GmbH (Germany) [12]. The H5 insert of the.