Many viral proteins are related to suppressing apoptosis in target cells and are hence beneficial to viral replication. The V protein of Newcastle disease virus (NDV) is one such protein that plays an important role in inhibiting apoptosis in a species-specific manner. However, to date, there have been no reports clarifying the antiapoptotic mechanisms of the V protein. The present study was undertaken to determine the apoptotic potential of the V protein in a chicken embryo fibroblast cell line (DF-1 cell) and to elucidate its molecular mechanisms of action. Here, a yeast two-hybrid system was used to screen the host proteins that interact with the V protein and identified thioredoxin-like protein 1 (TXNL1) as a potential binding partner. Immuno-colocalization of V protein and TXNL1 protein in DF-1 cells further verified the interaction of the two proteins. Through the overexpression of TXNL1 protein and knockdown of TXNL1 protein in DF-1 cells, the effects of NDV replication and cell apoptosis were examined. Cell apoptosis was detected by flow cytometry. The mRNA and protein expression levels of Bax, Bcl-2 and Caspase-3 were detected by quantitative real-time PCR (Q-PCR) and Western blotting. NDV expression was detected by Q-PCR and plaque assay. The results revealed that the TXNL1 protein induced apoptosis and inhibited NDV replication in DF-1 cells. Furthermore, the Western blot and Q-PCR results suggested that TXNL1 induced cell apoptosis through a pathway involving Bcl-2\Bax and Caspase-3. Finally, this work provides insight into the mechanism by which the V protein inhibits apoptosis.

Background: Newcastle disease (ND), which is caused by the Newcastle disease virus (NDV), is one of the most important avian diseases in poultry. Since its discovery in 1926, ND has caused great economic losses to the world poultry industry and remains a threat to chickens and wild birds. Although a stringent vaccination policy is widely adopted to control ND, ND outbreaks still occur, and virulent NDV is sporadically isolated from chickens and wild birds. To study the pathogenesis of ND and provide tools to prevent its prevalence, novel antibody fragments should be developed. The variable domains of the heavy chain of the heavy-chain antibodies (VHH) are the smallest naturally occurring antibodies derived from camelid heavy-chain antibodies. The comparatively small size, high affinity, high solubility, low immunogenicity and ability to bind epitopes inaccessible to conventional antibodies of VHH make them ideal candidates for a considerable number of therapeutic and biotechnological applications. However, an anti-NDV VHH has not been reported to date. Results: In this study, a VHH yeast two-hybrid library was constructed from NDV vaccine immunized C. bactrianus, and seven VHH fragments to the haemagglutinin-neuraminidase (HN) protein of NDV were successfully screened and characterized for the first time. These selected VHH clones were all expressed as soluble protein in E. coli. ELISA, dot blot, immunocytochemistry and pull down results showed that the screened VHHs could interact with NDV virion, among which five had neutralizing activity. In addition, the seven VHHs could inhibit the haemagglutination activity of different NDV strains. Conclusions: We constructed an NDV-immunized VHH yeast two-hybrid library and screened and characterized seven VHHs targeting NDV HN protein for the first time. The seven VHHs may have great potential for NDV diagnosis, pathogenesis and therapeutics.

Newcastle disease virus (NDV) can select cells to infect, but the mechanism of its cell selectivity has not been comprehensively investigated. Here, we use HeLa cells to establish that NDV can selectively infect cells at the single-cell level. We labeled proliferating cells with 5′-bromo-2-deoxyuridine (BrdU) and examined the colocalization of BrdU with NDV in cells to clarify the relationships between NDV infection and cell proliferation. Receptors at the plasma membrane mediate NDV entry into host cells. We labeled sialic acid receptor isoforms, compared their densities between different cell types and measured the sialic acid receptor densities in different cell phases. Our results suggest that NDV displays host tropism to HeLa cells compared to BHK cells and that the differences in the receptor isoform expression patterns between cell types contribute to the selection of HeLa by NDV. At the single-cell level, the dynamics of receptor expression changes during different cell phases contributing to the selection of cells in S/G2 phase for NDV infection. Furthermore, cell proliferation benefits viral replication, and enhanced virus replication leads to increased damage to cells. The elucidation of the mechanisms underlying host cell selection by NDV may help in the screening and characterizing of additional candidate oncolytic virus strains.

Contagious ecthyma is an acute infectious zoonosis caused by orf virus (ORFV). Live-attenuated ORFV vaccines have played a crucial role in preventing contagious ecthyma for decades. However, these vaccines often fail to induce long-lasting immunity. In recent years, numerous ORFV genome sequences have been published, yet genomic data for attenuated strains remain limited. Furthermore, no comprehensive whole-genome-based single nucleotide polymorphisms (SNPs) analysis has been conducted to compare ORFV wild-type and attenuated strains. In this study, we performed whole-genome sequencing of ORFV wild-type and attenuated strains from Shaanxi Province. We identified two ORFV strains with genomes shorter than 130 kb, which are closely related to the SC1 attenuated strain from Sichuan Province. Additionally, we noticed that 24 genes in the attenuated strain had SNPs, with the highest number of mutations occurring in the ORF022 gene. The function of the ORF022 gene has not been previously reported. Through in vitro experiments, we demonstrated that overexpression of ORF022 enhances ORFV replication in cells. The RNA-sequencing analysis revealed that ORF022 modulates host inflammation-related signaling pathways, as evidenced by the suppression of TNF, IL-17, and Toll-like receptor signaling pathways. Our findings suggest that the ORF022 in ORFV wild-type strain inhibits the host inflammatory response, reduces the immune response to ORFV, and facilitates viral replication. SNP events in attenuated strains (aFX0910) are one of the reasons for its attenuation. Investigations into the genomic sequences of attenuated viruses and the functional impact of mutated genes provide valuable insights into the mechanisms underlying ORFV attenuation and offer a foundation for the development of more effective ORFV vaccines.