Chimeric Newcastle disease virus-vectored vaccine protects chickens against H9N2 avian influenza virus in the presence of pre-existing NDV immunity.

A chimeric Newcastle disease virus (NDV) vector (NDV/AI4-TFHN) was constructed with the replacement of the ectodomains of the fusion and hemagglutinin-neuraminidase proteins by those from avian paramyxovirus type 2. The chimeric virus induced high antibody response in chickens pre-immunized with NDV. A recombinant vaccine candidate, NDV/AI4-TFHN-H9, expressing the hemagglutinin of H9N2 avian influenza virus, was generated, on the basis of the chimeric NDV vector mentioned above. The NDV/AI4-TFHN-H9 vaccine elicited H9-specific hemagglutination inhibition antibodies in chickens pre-immunized with NDV vaccine, and reduced the numbers of chickens shedding virus after H9N2 challenge. NDV/AI4-TFHN-H9 could serve as an alternative vaccine for the prevention of H9N2 infection in commercial poultry flocks.

MicroRNA Expression Profiling in Newcastle Disease Virus-Infected DF-1 Cells by Deep Sequencing.

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.

Gga-miR-19b-3p Inhibits Newcastle Disease Virus Replication by Suppressing Inflammatory Response via Targeting RNF11 and ZMYND11.

Newcastle disease (ND), an acute and highly contagious avian disease caused by virulent Newcastle disease virus (NDV), often results in severe economic losses worldwide every year. Although it is clear that microRNAs (miRNAs) are implicated in modulating innate immune response to invading microbial pathogens, their role in host defense against NDV infection remains largely unknown. Our prior study indicates that gga-miR-19b-3p is up-regulated in NDV-infected DF-1 cells (a chicken embryo fibroblast cell line) and functions to suppress NDV replication. Here we report that overexpression of gga-miR-19b-3p promoted the production of NDV-induced inflammatory cytokines and suppressed NDV replication, whereas inhibition of endogenous gga-miR-19b-3p expression had an opposite effect. Dual-luciferase and gene expression array analyses revealed that gga-miR-19b-3p directly targets the mRNAs of ring finger protein 11 (RNF11) and zinc-finger protein, MYND-type containing 11 (ZMYND11), two negative regulators of nuclear factor kappa B (NF-κB) signaling, in DF-1 cells. RNF11 and ZMYND11 silencing by small interfering RNA (siRNA) induced NF-κB activity and inflammatory cytokine production, and suppressed NDV replication; whereas ectopic expression of these two proteins exhibited an opposite effect. Our study provides evidence that gga-miR-19b-3p activates NF-κB signaling by targeting RNF11 and ZMYND11, and that enhanced inflammatory cytokine production is likely responsible for the suppression of NDV replication.

The PB2 and M genes of genotype S H9N2 virus contribute to the enhanced fitness of H5Nx and H7N9 avian influenza viruses in chickens.

Genotype S H9N2 viruses frequently donate their internal genes to facilitate the generation of novel influenza viruses, e.g., H5N6, H7N9, and H10N8, which have caused human infection. Genotype S was originated from the replacement of F/98-like M and PB2 genes of the genotype H with those from G1-like lineage. However, whether this gene substitution will influence the viral fitness of emerging influenza viruses remains unclear. We found that H5Nx and H7N9 viruses with G1-like PB2 or M gene exhibited higher virulence and replication than those with F/98-like PB2 or M in chickens. We also determined the functional significance of G1-like PB2 in conferring increased polymerase activity and improved nucleus transportation efficiency, and facilitated RNP nuclear export by G1-like M. Our results suggest that G1-like PB2 and M genes optimize viral fitness, and thus play a crucial role in the genesis of emerging influenza viruses that cause rising prevalence in chickens.