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Infectious bursal disease (IBD) is an avian viral disease that causes severe economic losses in the poultry industry worldwide. The live IBD virus (IBDV) has a potential immunosuppressive effect. Currently available IBDV vaccines have shortcomings, prompting the development of safer and more effective vaccination approaches, including the use of the recombinant turkey herpesvirus vaccine expressing the immunogenic structural VP2 protein of IBDV (recombinant HVT (rHVT)-IBD). The objectives of this study were twofold: (i) to develop in vitro assays and molecular tools to detect the VP2 protein and gene and (ii) to evaluate cell-mediated immunity (CMI) induced by rHVT-IBD vaccination of day-old specific pathogen-free chickens. The VP2 protein expressed by rHVT-IBD-infected chicken embryo fibroblasts was detected using the enzyme-linked immunosorbent assay and immunofluorescence. Using molecular techniques, the VP2 gene was detected in various organs, providing a method to monitor vaccine uptake. rHVT-IBD vaccination induced CMI responses in specific pathogen-free chickens at 5 weeks. CMI was detected by measuring chicken interferon-gamma after ex vivo antigenic stimulation of splenocytes. Moreover, our results showed that the enzyme-linked immunospot approach is more sensitive in detecting chicken interferon-gamma than enzyme-linked immunosorbent assay. The tools developed in this study may be useful in the characterization of new-generation recombinant vaccines and the cellular immune response they induce.

The G1-H9N2 avian influenza virus (AIV) has caused significant economic losses in the commercial poultry industry due to reduced egg production and increased mortality. The field observations have shown that H9N2 viruses circulate and naturally mix with other pathogens and these simultaneous infections can exacerbate disease. To avoid an incorrect virus characterization, due to co-infection, isolates were purified by in vitro plaque assays. Two plaque purified G1-H9N2 clones, selected on different cell types, named MDCK-and CEF-clone in regards to the cell culture used, were studied in vivo, revealing two different virulence phenotypes. Subsequently, the underlying mechanisms were studied. Specifically, the phenotypical outcome of SPF bird infection by the two clones resulted in completely different clinical outcomes. These differences in clinical outcome were used to study the factors behind this output in more detail. Further studies demonstrated that the more severe disease outcome associated with the MDCK-clone involves a strong induction of pro-inflammatory cytokines and a lack of type I interferon production, whereas the mild disease outcome associated with the CEF-clone is related to a greater antiviral cytokine response. The immunosuppressive effect of the MDCK-clone on splenocytes was further demonstrated via ChIFN-γ lack production after ex vivo mitogenic stimulation. Genome sequencing of the two clones identified only four amino acid differences including three in the HA sequence (HA-E198A, HA-R234L, HA-E502D-H9 numbering) and one in the NA sequence (NA-V33M). In the present study, valuable insights on the mechanisms responsible for AI pathogenicity and molecular mechanisms of H9N2 infections in chicken were obtained while highlighting the impact of the cells viruses are grown on their virulence.

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Recombinant Newcastle disease viruses (rNDV) have been used as bivalent vectors for vaccination against multiple economically important avian pathogens. NDV-vectored vaccines expressing the immunogenic H5 hemagglutinin (rNDV-H5) are considered attractive candidates to protect poultry from both highly pathogenic avian influenza (HPAI) and Newcastle disease (ND). However, the impact of the insertion of a recombinant protein, such as H5, on the biological characteristics of the parental NDV strain has been little investigated to date. The present study compared a rNDV-H5 vaccine and its parental NDV LaSota strain in terms of their structural and functional characteristics, as well as their recognition by the innate immune sensors. Structural analysis of the rNDV-H5 demonstrated a decreased number of fusion (F) and a higher number of 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.

16To overcome some of the shortcomings of classical whole virus avian influenza (AI) inactivated 17 vaccines, recombinant vector vaccines, such as recombinant Newcastle disease virus (NDV) 18 vaccines expressing the immunogenic H5 hemagglutinin of AI (rNDV-H5), have been 19 developed. The impact of H5 insertion and surface expression on NDV structure and 20 functionality of a specific bivalent rNDV-H5 vaccine was investigated here. 21Structural analysis by immunogold labeling demonstrated the impact of the H5 expression on the 22 was observed compared with the parental strain, confirming the H5-involvement in the rNDV-32 H5 entry. 33 IMPORTANCE To prevent economic loss in the poultry industry, vaccination against two 34 major treats, Newcastle Disease Virus (NDV) and Avian Influenza Virus (AIV), is one of major 35 strategy. However, despite intense vaccination campaigns, ND continues to circulate, and AI 36 vaccination expresses shortcomings. In consequence, bivalent vaccines improving 37 simultaneously AI and ND protection in poultry are developed, and tested for their protective 38 hybrid apathogenic clade 1 HP H5 hemagglutinin (rNDV-H5), developed at the Mount Sinai 60 School of Medicine based on their rNDV/F3aa-chimericH7 work (10), has demonstrated its 61 efficacy in SPF chickens against both homologous and heterologous HPAI H5 (11-14) and 62 velogenic ND challenges (13). 63The H5-expression at the surface of rNDV-H5 virions of this experimental vaccin has previously 64 been confirmed by immunofluorescence (11) and immunogold electron microscopy (11, 15). 65However, the impact of the H5 insertion on the ND virus (NDV) surface glycoprotein 66 assessing the potential impact of the expression of H5 on the vector binding, entry and release 84 processes. 85 RESULTS 86 Evaluation of rNDV-H5 glycoproteins expression 87Identical nucleotide sequences were observed between the hybrid H5 and the native H5 from 126 HPAI H5N1 clade 1, apart from the engineered attenuated cleavage site and the NDV F-replaced 127 CT and TM domain of the hybrid H5. 128For the HN sequence a unique mutation was detected in the HN from rNDV-H5, leading to a 129 S521R change in its amino acid sequence compared to the NDV LaSota HN. 130 DISCUSSION 131 8The rNDV-H5-vaccine, obtained by reverse genetics from a common LaSota vaccine strain, 132