Cellular MicroRNA Expression Profile of Chicken Macrophages Infected with Newcastle Disease Virus Vaccine Strain LaSota.
ABSTRACT: Vaccines with live, low-virulence Newcastle disease virus (NDV) strains are still the most accepted prevention and control strategies for combating Newcastle disease (ND), a major viral disease that hampers the development of the poultry industry worldwide. However, the mechanism underlying vaccine-mediated innate cell immune responses remains unclear. Here, a high-throughput Illumina sequencing approach was employed to determine cellular miRNA expression profiles in chicken macrophages infected with the LaSota virus, a widely used vaccine strain for mass vaccination programs against ND in poultry. Compared to the control group, 112 and 115 differentially expressed (DE) miRNAs were identified at 24 hpi (hours post inoculation) and 48 hpi, respectively. Meanwhile, 174 DE miRNAs were identified between 24 hpi and 48 hpi. Furthermore, 12 upregulated and 6 downregulated DE miRNAs were observed in common at 24 and 48 hpi compared with 0 hpi. In addition, target prediction and functional analysis of these DE miRNAs revealed significant enrichment for several signaling pathways, especially in the immune-related genes and pathways, such as the RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, and mitogen-activated protein kinase (MAPK) signaling pathway. Our findings not only lay the foundations for further investigating the roles and regulatory mechanisms of miRNA in vaccine-mediated innate cellular immune responses, but also extend new insights into the interactions between the host and NDV infection.
Project description:Infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) are two poultry pathogens seriously affecting the poultry industry. Here, IBV S1 and the ectodomain of NDV F proteins were separately linked with the trans-membrane and carboxy-terminal domain of IBV S protein (STMCT), composing rS and rF; thus, a novel chimeric infectious bronchitis-Newcastle disease (IB-ND) virus-like particles (VLPs) vaccine containing the rS, rF, and IBV M protein was constructed. Under the transmission electron microscope (TEM), VLPs possessing similar morphology to natural IBV were observed. To evaluate the immunogenicity of chimeric IB-ND VLPs, specific pathogen-free (SPF) chickens were immunized with three increasing doses (50, 75, and 100 ?g protein of VLPs). Results of ELISAs detecting IBV and NDV specific antibodies and IL-4 and IFN-? T cell cytokines indicated that vaccination with chimeric IB-ND VLPs could efficiently induce humoral and cellular immune responses. In the challenge study, chimeric IB-ND VLPs (100 ?g protein) provided 100% protection against IBV or NDV virulent challenge from death, and viral RNA levels in tissues and swabs were greatly reduced. Collectively, chimeric IB-ND VLPs are highly immunogenic and could provide complete protection from an IBV or NDV virulent challenge. Chimeric IB-ND VLPs are an appealing vaccine candidate and a promising vaccine platform bearing multivalent antigens.
Project description:Newcastle disease (ND) is one of the most important diseases of poultry, negatively affecting poultry production worldwide. The disease is caused by Newcastle disease virus (NDV) or avian paramyxovirus type 1 (APMV-1), a negative-sense single-stranded RNA virus of the genus Avulavirus, family Paramyxoviridae. Although all NDV isolates characterized to date belong to a single serotype of APMV-1, significant genetic diversity has been described between different NDV isolates. Here we present the complete genome sequence and the clinicopathological characterization of a virulent Newcastle disease virus isolate (NDV-Peru/08) obtained from poultry during an outbreak of ND in Peru in 2008. Phylogenetic reconstruction and analysis of the evolutionary distances between NDV-Peru/08 and other isolates representing established NDV genotypes revealed the existence of large genomic and amino differences that clearly distinguish this isolate from viruses of typical NDV genotypes. Although NDV-Peru/08 is a genetically distinct virus, pathogenesis studies conducted with chickens revealed that NDV-Peru/08 infection results in clinical signs characteristic of velogenic viscerotropic NDV strains. Additionally, vaccination studies have shown that an inactivated NDV-LaSota/46 vaccine conferred full protection from NDV-Peru/08-induced clinical disease and mortality. This represents the first complete characterization of a virulent NDV isolate from South America.
Project description:Newcastle disease virus (NDV) and Salmonella Pullorum have significant damaging effects on the poultry industry, but no previous vaccine can protect poultry effectively. In this study, a recombinant-attenuated S. Pullorum strain secreting the NDV hemagglutinin-neuraminidase (HN) protein, C79-13?crp?asd (pYA-HN), was constructed by using the suicide plasmid pREasd-mediated bacteria homologous recombination method to form a new bivalent vaccine candidate against Newcastle disease (ND) and S. Pullorum disease (PD). The effect of this vaccine candidate was compared with those of the NDV LaSota and C79-13?crp?asd (pYA) strains. The serum hemagglutination inhibition antibody titers, serum immunoglobulin G (IgG) antibodies, secretory IgA, and stimulation index in lymphocyte proliferation were increased significantly more (p < 0.01) in chickens inoculated with C79-13?crp?asd (pYA-HN) than with C79-13?crp?asd (pYA) but were not significantly increased compared with the chickens immunized with the LaSota live vaccine (p > 0.05). Moreover, the novel strain provides 60% and 80% protective efficacy against the NDV virulent strain F48E9 and the S. Pullorum virulent strain C79-13. In summary, in this study, a recombinant-attenuated S. Pullorum strain secreting NDV HN protein was constructed. The generation of the S. Pullorum C79-13?crp?asd (pYA-HN) strain provides a foundation for the development of an effective living-vector double vaccine against ND and PD.
Project description:Newcastle disease virus (NDV), causative agent of Newcastle disease (ND), is one of the most devastating pathogens for poultry industry worldwide. MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by regulating mRNA translation efficiency or mRNA abundance through binding to mRNA directly. Accumulating evidence has revealed that cellular miRNAs can also affect virus replication by controlling host-virus interaction. To identify miRNA expression profile and explore the roles of miRNA during NDV replication, in this study, small RNA deep sequencing was performed of non-inoculated DF-1 cells (chicken embryo fibroblast cell line) and JS 5/05-infected cells collected at 6 and 12 h post infection (hereafter called mock' NDV-6 h, and NDV-12 h groups respectively). A total of 73 miRNAs of NDV-6 h group and 64miRNAs of NDV-12 h group were significantly differentially expressed (SDE) when compared with those in mock group. Meanwhile, 50 SDE miRNAs, including 48 up- and 2 down-regulated, showed the same expression patterns in NDV-6 h and NDV-12 h groups. qRT-PCR validation of 15 selected miRNAs' expression patterns was consistent with deep sequencing. To investigate the role of these SDE miRNAs in NDV replication, miRNA mimics and inhibitors were transfected into DF-1 cells followed by NDV infection. The results revealed that gga-miR-451 and gga-miR-199-5p promoted NDV replication while gga-miR-19b-3p and gga-miR-29a-3p inhibited NDV replication. Further function research demonstrated gga-miR-451 suppressed NDV-induced inflammatory response via targeting YWHAZ (tyrosine3-monooxygenase/tryptophan5-monooxygenase activation protein zeta). Overall, our study presented a global miRNA expression profile in DF-1 cells in response to NDV infection and verified the roles of some SDE miRNAs in NDV replication which will underpin further studies of miRNAs' roles between the host and the virus.
Project description:Newcastle disease (ND), caused by virulent Newcastle disease virus (NDV) strains, has been one of the most problematic diseases affecting the poultry industry worldwide. Conventional vaccines provide effective protection for birds to survive ND outbreaks, but they may not completely suppress NDV shedding. NDV strains circulate on farms for a long time after the initial infection and cause potential risks. A new vaccine with fast clearance ability and low viral shedding is needed. In this study, we used interleukin-12 (IL-12) as an adjuvant and electroporation (EP) as an advanced delivery system to improve a DNA vaccine candidate. The fusion (F) protein gene from an NDV strain of the prevalent genotype VII.1.1 was cloned to prepare the vaccine. Chickens immunized with the F gene DNA vaccine co-delivered with an IL-12-expressing plasmid DNA showed higher neutralizing antibody levels and stronger concanavalin-A-induced lymphocyte proliferation than those treated with the F gene DNA vaccine alone. The co-delivered vaccine provided 100% protection, and less viral shedding and a shorter release time were observed in challenged chickens than when the F gene DNA vaccine was administered alone. The use of F gene DNA combined with IL-12 delivered by electroporation is a promising approach for vaccination against ND.
Project description:Newcastle disease (ND) is one of the most devastating poultry infections because of its worldwide distribution and accompanying economical threat. In the present study, we characterized the ND virus (NDV) K148/08 strain from wild mallard duck, with regard to safety, thermostability, immunogenicity, and protective efficacy against velogenic ND viral infection. The NDV K148/08 strain offered enhanced immunogenicity and safety relative to commercially available vaccine strains. The NDV K148/08 strain was safe in 1-day-old SPF chicks after vaccination using a coarse or cabinet-type fine sprayer. We demonstrated that the NDV K148/08 strain elicited high levels of antibody responses and provided protective efficacy against lethal NDV challenge. In addition, the thermostability of the NDV K148/08 strain was as high as that of the thermostable V4 strain. Therefore, the NDV K148/08 strain may be useful to ensure NDV vaccine performance and effectiveness in developing countries, especially in remote areas without cold chains.
Project description:Highly pathogenic avian influenza (HPAI) and Newcastle disease (ND) are considered as the most devastating poultry infections, owing to their worldwide distribution and economical threat. Vaccines have been widely used to control these diseases in the poultry industry in endemic countries. However, vaccination policy without differentiating infected animals from vaccinated animals (DIVA) makes the virus surveillance difficult. In this study, we developed a bivalent virus-like particle (VLP) vaccine that is composed of the hemagglutinin (HA) and matrix 1 (M1) proteins of the H5N1 HPAI virus (HPAIV) and a chimeric protein containing the ectodomain of the ND virus (NDV) fusion (F) protein fused with the cytoplasmic and transmembrane domains of the HPAIV HA protein. A single immunization of chickens with the chimeric VLP vaccine induced high levels of hemagglutination inhibition (HI) antibody titers against H5N1 HPAI virus and anti-NDV antibody detected in ELISA and protected chickens against subsequent lethal HPAIV and NDV infections. Furthermore, we could easily perform DIVA test using the commercial NP-cELISA tests against HPAIV and HI assay against NDV. These results strongly suggest that utilization of chimeric VLP vaccine in poultry species would be a promising strategy for the better control of HPAI and ND simultaneously.
Project description:Newcastle disease (ND) is a serious disease of poultry that causes significant economic losses. Despite rampant ND outbreaks that occur annually in Kenya, the information about the NDV circulating in Kenya is still scarce. We report the first countrywide study of NDV in Kenya. Our study is aimed at evaluating the genetic characteristics of Newcastle disease viruses obtained from backyard poultry in farms and live bird markets in different regions of Kenya. We sequenced and analyzed fusion (F) protein gene, including the cleavage site, of the obtained viruses. We aligned and compared study sequences with representative NDV of different genotypes from GenBank. The fusion protein cleavage site of all the study sequences had the motif 112RRQKRFV118 indicating their velogenic nature. Phylogenetic analysis revealed that the NDV from various sites in Kenya was highly similar genetically and that it clustered together with NDV of genotype V. The study samples were 96% similar to previous Ugandan and Kenyan viruses grouped in subgenotype Vd This study points to possible circulation of NDV of similar genetic characteristics between backyard poultry farms and live bird markets in Kenya. The study also suggests the possible spread of velogenic NDV between Kenya and Uganda possibly through cross-border live bird trade. Our study provides baseline information on the genetic characteristics of NDV circulating in the Kenyan poultry population. This highlights the need for the ND control programmes to place more stringent measures on cross-border trade of live bird markets and poultry products to prevent the introduction of new strains of NDV that would otherwise be more difficult to control.
Project description:The highly contagious Newcastle disease virus (NDV) continues to threaten poultry all over the world. The NDV DNA vaccine is a promising solution to the current Newcastle disease (ND) challenges, and thus an efficient delivery system should be developed to facilitate the efficacy of DNA vaccines. In this study, we developed a DNA vaccine delivery system consisting of a triblock copolymer of poly(lactide co-glycolide acid) and polyethylene glycol (PLGA-PEG-PLGA) hydrogel in which the recombinant NDV hemagglutinin-neuraminidase (HN) plasmid was encapsulated. Its characteristics, security, immune responses, and efficacy against highly virulent NDV were detected. The results showed that the plasmids were gradually released in a sustained manner from the hydrogel, which improved the biological stability of the plasmids and demonstrated a high biocompatibility. The plasmids, when they were incorporated into the hydrogel delivery system, enhanced immune activation and provided 100% protection against the highly virulent NDV strain. Furthermore, we proved that this NDV DNA hydrogel vaccine could improve the lymphocyte proliferation and increase the immunological cytokine production via the PI3K/Akt pathway. These results indicate that the PLGA-PEG-PLGA thermosensitive hydrogel could be a promising delivery system for the NDV DNA vaccine in order to achieve a sustained supply of plasmids and induce potent immune responses.
Project description:BACKGROUND:Newcastle Disease (ND) is a highly contagious and economically devastating disease of poultry. At present, limited molecular epidemiological data are available regarding the causes of ND outbreaks in vaccinated commercial poultry farms. Knowing the genomic characteristics of Newcastle disease virus (NDV) infecting commercial poultry operations in spite of vaccination might give important insights on the infection dynamics of these viruses. In addition, molecular analyses at the subgenotype level and studies on the relationship of Japanese NDVs with other isolates from around the world are lacking. Therefore, in the present study, a molecular epidemiological investigation was conducted to characterize nine NDVs isolated from vaccinated commercial poultry flocks in five different Prefectures in non-epidemic areas of Japan between 1969 and 2002. METHODS:Nucleotide sequencing and phylogenetic studies were performed to characterize the complete fusion (F)-protein gene, 3-prime end of the nucleoprotein (NP)-gene and 5-prime end of the RNA dependent RNA polymerase (L)-gene. Sequence data were compared with 180 NDV strains from GenBank representing different NDV genotypes and subgenotypes from different regions of the world at different time periods. Deduced amino acids were analyzed for homologies, recombination and mutation. Recombination events were estimated using Recombination Detection Program (RDP) version 3.44. Phylogenetic trees were constructed to determine evolutionary relationships among strains. RESULTS:Mean death time (MDT: 48-56 hr), Intracerebral Pathogenicity Index (ICPI: 1.7-1.9) and deduced amino acid sequences of the F0 proteolytic cleavage site (112RRQKR116) revealed that all nine field isolates were velogenic. Phylogenetic analysis showed that these isolates could be classified into two genetic lineages and three sublineages namely genotypes VIa (lineage 4a), VId (lineage 4d) and VIId (lineage 5d). No recombination events were observed but a point mutation in one of the neutralizing epitope of the F-protein was identified in the field isolates from Japan. CONCLUSIONS:All field isolates from vaccinated commercial poultry in non-epidemic areas of Japan were part of much bigger outbreaks in provinces and regions and, in some cases, continents. In general, four ND panzootics occurred in Japan and that these outbreaks were mostly characterized by co-circulation of genetically distinct virus lineages due to involvements of infected wild birds. The point mutation identified in the field isolates from Japan may be due to escape from vaccine pressure. The identification of such mutation may be useful for future site-directed mutagenesis to understand the dynamics of NDV infection in vaccinated chickens.