Classical swine fever virus (CSFV), the pathogen of classical swine fever (CSF), causes severe hemorrhagic fever and vascular necrosis in domestic pigs and wild boar. A large number of evidence has proven that non-structural 5A (NS5A) is not only a very important part of viral replication complex, but also can regulate host cell’s function; however, the underlying mechanisms remain poorly understood. In the current study, aiming to find more clues in understanding the molecular mechanisms of CSFV NS5A’s function, the yeast two-hybrid (Y2H) system was adopted to screen for CSFV NS5A interactive proteins in the cDNA library of the swine umbilical vein endothelial cell (SUVEC). Alignment with the NCBI database revealed 16 interactive proteins: DDX5, PSMC3, NAV1, PHF5A, GNB2L1, CSDE1, HSPA8, BRMS1, PPP2R3C, AIP, TMED10, POLR1C, TMEM70, METAP2, CHORDC1 and COPS6. These proteins are mostly related to gene transcription, protein folding, protein degradation and metabolism. The interactions detected by the Y2H system should be considered as preliminary results. Since identifying novel pathways and host targets, which play essential roles during infection, may provide potential targets for therapeutic development. The finding of proteins obtained from the SUVEC cDNA library that interact with the CSFV NS5A protein provide valuable information for better understanding the interactions between this viral protein and the host target proteins.

The use of adenovirus vector-based vaccines is a promising approach for generating antigen-specific immune responses. Improving vaccine potency is necessary in other approaches to address their inadequate protection for the majority of infectious diseases. This study is the first to reconstruct a recombinant replication-defective human adenovirus co-expressing E2 and invasin C-terminal (InvC) glycoproteins (rAd-E2-InvC). rAd-E2-InvC with 2×10⁶ TCID₅₀ was intramuscularly administered two times to CSFV-free pigs at 14 day intervals. No adverse clinical reactions were observed in any of the pigs after the vaccination. The CSFV E2-specific antibody titer was significantly higher in the rAd-E2-InvC group than that in the rAdV-E2 group as measured by NPLA and blocking ELISA. Pigs immunized with rAd-E2-InvC were completely protected against lethal challenge. Neither CSFV RNA nor pathological changes were detected in the tissues after CSFV challenge. These results demonstrate that rAd-E2-InvC could be an alternative to the existing CSF vaccine. Moreover, InvC that acts as an adjuvant could enhance the immunogenicity of rAdV-E2 and induce high CSFV E2-specific antibody titer and protection level.

Porcine circovirus type 2 (PCV2) is the primary infectious agent of PCV-associated disease (PCVAD) in swine. ORF4 protein is a newly identified viral protein of PCV2 and is involved in virus-induced apoptosis. However, the molecular mechanisms of ORF4 protein regulation of apoptosis remain unclear, especially given there is no information regarding any cellular partners of the ORF4 protein. Here, we have utilized the yeast two-hybrid assay and identified four host proteins (FHC, SNRPN, COX8A and Lamin C) interacting with the ORF4 protein. Specially, FHC was chosen for further characterization due to its important role in apoptosis. GST pull-down, subcellular co-location and co-immunoprecipitation assays confirmed that the PCV2 ORF4 protein indeed interacted with the heavy-chain ferritin, which is an interesting clue that will allow us to determine the role of the ORF4 protein in apoptosis.

The Golgi apparatus and its resident proteins are utilized and regulated by viruses to facilitate their proliferation. Inthis study, we investigated Classical swine fever virus (CSFV) proliferation when the function of the Golgi wasdisturbed. Golgi function was disturbed using chemical inhibitors, namely, brefeldin A (BFA) and golgicide A (GCA),and RNA interfering targets, such as the Golgi-specific BFA-resistance guanine nucleotide exchange factor 1 (GBF1)and Rab2 GTPases. CSFV proliferation was significantly inhibited during RNA replication and viral particlegeneration after BFA and GCA treatment. CSFV multiplication dynamics were retarded in cells transfected withGBF1 and Rab2 shRNA. Furthermore, CSFV proliferation was promoted by GBF1 and Rab2 overexpression using alentiviral system. Hence, Golgi function is important for CSFV multiplication, and GBF1 and Rab2 participate inCSFV proliferation. Further studies must investigate Golgi-resident proteins to elucidate the mechanism underlyingCSFV replication.

Classical swine fever (CSF) is a contagious disease with a high mortality rate and is caused by classical swine fever virus(CSFV). CSFV non-structural protein 4B (NS4B) plays a crucial role in CSFV replication and pathogenicity. However,precisely how NS4B exerts these functions remains unknown, especially as there are no reports relating to potential cellularpartners of CSFV NS4B. Here, a yeast two-hybrid (Y2H) system was used to screen the cellular proteins interacting withNS4B from a porcine alveolar macrophage (PAM) cDNA library. The protein screen along with alignment using the NCBIdatabase revealed 14 cellular proteins that interact with NS4B: DDX39B, COX7C, FTH1, MAVS, NR2F6, RPLP1,PSMC4, FGL2, MKRN1, RPL15, RPS3, RAB22A, TP53BP2 and TBK1. These proteins mostly relate to oxidoreductaseactivity, signal transduction, localization, biological regulation, catalytic activity, transport and metabolism by GO categories.Tank-binding kinase 1 (TBK1) was chosen for further confirmation. The NS4B-TBK1 interaction was furtherconfirmed by subcellular co-location, co-immunoprecipitation and glutathione S-transferase pull-down assays. This studyoffers a theoretical foundation for further understanding of the diversity of NS4B functions in relation to viral infection andsubsequent pathogenesis.