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Calves were infected intranasally and intratracheally with Newcastle disease virus (NDV), an avian paramyxovirus. Clinical signs, viral replication, and antibody production were evaluated. This study showed that NDV replicated in calves, as evidenced by development of NDV-specific humoral and mucosal antibody responses, but was attenuated in this unnatural host. These results suggest that NDV has the potential for development as a host-range-restricted, intranasal vaccine vector for cattle that lack preexisting immunity to NDV.

Newcastle disease virus (NDV), a member of the family Paramyxoviridae , has a nonsegmented negative-sense RNA genome consisting of six genes (3′-NP-P-M-F-HN-L-5′). The first three 3′-end intergenic sequences (IGSs) are single nucleotides (nt), whereas the F-HN and HN-L IGSs are 31 and 47 nt, respectively. To investigate the role of IGS length in NDV transcription and pathogenesis, we recovered viable viruses containing deletions or additions in the IGSs between the F and HN and the HN and L genes. The IGS of F-HN was modified to contain an additional 96 nt or more or a deletion of 30 nt. Similarly, the IGS of HN-L was modified to contain an additional 96 nt or more or a deletion of 42 nt. The level of transcription of each mRNA species (NP, F, HN, and L) was examined by Northern blot analysis. Our results showed that NDV can tolerate an IGS length of at least 365 nt. The extended lengths of IGSs down-regulated the transcription of the downstream gene and suggested that 31 nt in the F-HN IGS and 47 nt in the HN-L IGS are required for efficient transcription of the downstream gene. The effect of IGS length on pathogenicity of mutant viruses was evaluated in embryonated chicken eggs, 1-day-old chicks, and 6-week-old chickens. Our results showed that all IGS mutants were attenuated in chickens. The level of attenuation increased as the length of the IGS increased. Interestingly, decreased IGS length also attenuated the viruses. These findings can have significant applications in NDV vaccine development.

To determine the role of amino acid sequences of the hemagglutinin-neuraminidase (HN) cytoplasmic tail in Newcastle disease virus (NDV) replication and pathogenicity, we generated recombinant NDVs with a deletion or point mutation in the N-terminal cytoplasmic tail. The first 2-amino-acid deletion in the cytoplasmic tail did not affect the biological characteristics of NDV. However, a 4-amino-acid deletion and the substitution of alanine for serine at position 6 affected cell fusion, pathogenicity, and colocalization of the HN and M proteins of NDV, indicating that these residues of the HN cytoplasmic tail are critical for its specific incorporation into virions.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a multifunctional protein that plays a crucial role in virus infectivity. In this study, using the mesogenic strain Beaudette C (BC), we mutated three conserved amino acids thought to be part of the binding/catalytic active site in the HN protein. We also mutated five additional residues near the proposed active site that are nonconserved between BC and the avirulent strain LaSota. The eight recovered NDV HN mutants were assessed for effects on biological activities. While most of the mutations had surprisingly little effect, mutation at conserved residue Y526 reduced the neuraminidase, receptor binding, and fusion activities and attenuated viral virulence in eggs and young birds.

To determine the role of untranslated regions (UTRs) in replication and pathogenesis of Newcastle disease virus (NDV), we generated recombinant viruses with deletions in 5′ and 3′ UTRs of the HN mRNA. Deletion of any HN UTR did not noticeably affect in vitro replication of these viruses. However, complete deletion of the 5′ UTR of the HN gene decreased the HN mRNA levels and HN protein contents in virus particles, resulting in attenuation of the virus in chickens. This indicates that the 5′ UTR of HN mRNA plays an important role in replication and pathogenicity of NDV in vivo.

The neonatal Fc receptor for IgG (FcRn) transfers maternal IgG to the offspring and protects IgG from degradation. The FcRn resides in an acidic intracellular compartment, allowing it to bind IgG. In this study, we found the association of FcRn and invariant chain (Ii). The interaction was initiated within the endoplasmic reticulum by Ii binding to either the FcRn H chain alone or FcRn H chain-β2-microglobulin complex and appeared to be maintained throughout the endocytic pathway. The CLIP in Ii was not required for FcRn-Ii association. The interaction was also detected in IFN-γ-treated THP-1, epithelial and endothelial cells, and immature mouse DCs. A truncated FcRn without the cytoplasmic tail was unable to traffic to early endosomes; however, its location in early endosomes was restored by Ii expression. FcRn was also detected in the late endosome/lysosome only in the presence of Ii or on exposure to IFN-γ. In immature human or mouse DCs, FcRn was barely detected in the late endosome/lysosome in the absence of Ii. Furthermore, the cytoplasmic tail of Ii conferred tailless FcRn to route to both the early endosome and late endosome/lysosome in a hybrid molecule. Because the FcRn is expressed in macrophages and DCs or epithelial and endothelial cells where Ii is induced under inflammation and infection, these results reveal the complexity of FcRn trafficking in which Ii is capable of expanding the boundary of FcRn trafficking. Taken together, the intracellular trafficking of FcRn is regulated by its intrinsic sorting information and/or an interaction with Ii chain.

Transcription activator-like effector nucleases (TALEN) are engineered proteins used for precise genome editing by generating specific DNA double strand that are repaired by homologous recombination and by non-homologous end joining. TALENs can be used to study gene regulation by deleting putative regulatory elements in the context of the native chromosome and measuring mRNA synthesis. We designed TALENs to delete individual DNAse I-hypersensitive sites (HS) of the β-globin locus control region (LCR) followed by an assessment of globin gene expression and assessment of epigenetic effects in K562 erythroleukemia cells. The β-globin LCR is composed of five HSs and functions as a powerful regulatory element responsible for appropriate levels of the five β-like globin genes during development. Introduction of plasmid DNA encoding a pair of TALENs and targeting individually the flanking region of the HS2, HS3 and HS4 core elements along with a donor 100 base single-stranded oligonucleotide resulted in the successful deletions of each of the three core elements in K562 cells. Individual K562 cells were seeded to produce clones and the mutations were screened by PCR to identify both heterozygous and homozygous clones. The TALEN-mediated 288 bp HS2 core deletion resulted 32 heterozygous (48.5%) and 6 homozygous clones (9.1%) in a total of 66 clones screened. K562 carries three copies of chromosome 11 emphasizing the robustness of TALEN technology to target each of the alleles. In the 199 bp HS3 core deletion, from 113 clones we identified 28 heterozygous (24.8%) and 3 (2.7%) homozygous clones. Lastly, the 301 bp HS4 core deletion yielded 9 homozygous (5.9%) and 12 heterozygous (7.9%) clones from 151 clones screened. Total RNA was isolated from wild-type K562 cells, and from both the heterozygous and homozygous mutant clones and subjected to RNase Protection analysis to quantitate the levels of globin mRNA. Deletion if the HS3 core in K562 cells in a ∼30% reduction in ε-globin mRNA and 2-fold reduction in γ-globin mRNA. A more dramatic effect on globin expression is observed in the HS2 core deletion, as ε- and γ-globin expression is reduced by 2- and 5-fold, respectively. These results suggest that HS2 contributes the majority of the LCR enhancer function in K562 cells. The HS4 core deletion resulted in a modest ∼20% reduction in both ε- and γ-globin expression. TALENs were designed to knockout trans-acting factors implicated to be involved in globin gene regulation and/or globin switching. TALENs bracketing the gene promoters and the first exon of 25 genes encoding either a transcription factor or histone-modifying enzyme were synthesized and post-transfection PCR screens of the transfected pool of K562 cells resulted in the successful identification of 17 gene knockouts. The 17 target genes are PRMT5, LDB1, EIF2AK3, BCL11A, HBSIL, MYB, SOX6, NFE4, NR2F2, NR2C1, NR2C2, CHTOP, NFE2, DNMT3A, RBBP4, MTA2 and MBD2. Single cell clones have been generated by limited dilution of transfected K562 pools and thus far we have identified heterozygous and homozygous clones of 8 of 17 gene knockouts, importantly all clones were identified without selection. The frequency of identifying the knockout clones, represented by the number of clones screened/ number of heterozygous clones/ number of homozygous clones, are as follows: HBS1L (63/3/0), SOX6 (68/13/2), NFE4 (56/13/7), LBD1 (300/2/0), MBD2 (301/0/1), CHTOP (288/66/6), NFE2 (712/44/5) and NR2C1 (96/40/11). The remaining nine gene knockouts and globin gene expression data will be presented at the meetings. These studies highlight a powerful TALEN-mutagenesis platform for target deletions of both cis- and trans-elements to study globin gene switching. TALENs can be synthesized in several days and the screening of the individual clones for the desired knockouts is completed within two weeks. This highly efficient mutagenesis platform will further our understanding of the molecular basis of globin switching. Disclosures: No relevant conflicts of interest to declare.