Porcine deltacoronavirus (PDCoV), first identified in 2012, is a swine enteropathogen now found in many countries. The nucleocapsid (N) protein, a core component of PDCoV, is essential for virus replication and is a significant candidate in the development of diagnostics for PDCoV. In this study, monoclonal antibodies (mAbs) were generated and tested for reactivity with three truncations of the full protein (N1, N2, N3) that contained partial overlaps; of the five monoclonals chosen tested, each reacted with only the N3 truncation. The antibody designated 4E88 had highest binding affinity with the N protein and was chosen for in-depth examination. The 4E88 epitope was located to amino acids 308-AKPKQQKKPKK-318 by testing the 4E88 monoclonal for reactivity with a series of N3 truncations, then the minimal epitope, 309-KPKQQKKPK-317 (designated EP-4E88), was pinpointed by testing the 4E88 monoclonal for reactivity with a series of synthetic peptides of this region. Homology analysis showed that the EP-4E88 sequence is highly conserved among PDCoV strains, and also shares high similarity with sparrow coronavirus (HKU17), Asian leopard cat coronavirus (ALCCoV), quail coronavirus (UAE-HKU30), and sparrow deltacoronavirus (SpDCoV). Of note, the PDCoV EP-4E88 sequence shared very low similarity (<22.2%) with other porcine coronaviruses (PEDV, TGEV, PRCV, SADS-CoV, PHEV), demonstrating that it is an epitope that can be used for distinguishing PDCoV and other porcine coronavirus. 3D structural analysis revealed that amino acids of EP-4E88 were in close proximity and may be exposed on the surface of the N protein.

The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus prevalent in east and southeast Asia, the Western Pacific, and northern Australia. Since viruses are obligatory intracellular pathogens, the dynamic processes of viral entry, replication, and assembly are dependent on numerous host-pathogen interactions. Efforts to identify JEV-interacting host factors are ongoing because their identification and characterization remain incomplete. Three enzymatic activities of flavivirus non-structural protein 3 (NS3), including serine protease, RNA helicase, and triphosphatase, play major roles in the flaviviruses lifecycle. To identify cellular factors that interact with NS3, we screened a human brain cDNA library using a yeast two-hybrid assay, and identified eight proteins that putatively interact with NS3: COPS5, FBLN5, PPP2CB, CRBN, DNAJB6, UBE2N, ZNF350, and GPR137B. We demonstrated that the DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6) colocalizes and interacts with NS3, and has a negative regulatory function in JEV replication. We also show that loss of DNAJB6 function results in significantly increased viral replication, but does not affect viral binding or internalization. Moreover, the time-course of DNAJB6 disruption during JEV infection varies in a viral load-dependent manner, suggesting that JEV targets this host chaperone protein for viral benefit. Deciphering the modes of NS3-interacting host proteins functions in virion production will shed light on JEV pathogenic mechanisms and may also reveal new avenues for antiviral therapeutics.

PDCoV is an emerging enteropathogenic coronavirus that mainly causes acute diarrhea in piglets, seriously affecting pig breeding industries worldwide. To date, the molecular mechanisms of PDCoV-induced immune and inflammatory responses or host responses in LLC-PK cells in vitro are not well understood. HSP90 plays important roles in various viral infections. In this study, HSP90AB1 knockout cells (HSP90AB1^KO) were constructed and a comparative transcriptomic analysis between PDCoV-infected HSP90AB1^WT and HSP90AB1^KO cells was conducted using RNA sequencing to explore the effect of HSP90AB1 on PDCoV infection. A total of 1295 and 3746 differentially expressed genes (DEGs) were identified in PDCoV-infected HSP90AB1^WT and HSP90AB1^KO cells, respectively. Moreover, most of the significantly enriched pathways were related to immune and inflammatory response-associated pathways upon PDCoV infection. The DEGs enriched in NF-κB pathways were specifically detected in HSP90AB1^WT cells, and NF-κB inhibitors JSH-23, SC75741 and QNZ treatment reduced PDCoV infection. Further research revealed most cytokines associated with immune and inflammatory responses were upregulated during PDCoV infection. Knockout of HSP90AB1 altered the upregulated levels of some cytokines. Taken together, our findings provide new insights into the host response to PDCoV infection from the transcriptome perspective, which will contribute to illustrating the molecular basis of the interaction between PDCoV and HSP90AB1.

The potD gene, belonging to the well-conserved ABC (ATP-binding cassette) transport system potABCD, encodes the bacterial substrate-binding subunit of the polyamine transport system. In this study, we found PotD in Haemophilus (Glaesserella) parasuis could actively stimulate both humoral immune and cellular immune responses and elevate lymphocyte proliferation, thus eliciting a Th1-type immune response in a murine immunity and infection model. Stimulation of Raw 264.7 macrophages with PotD validated that Toll-like receptor 4, rather than 2, participated in the positive transcription and expression of pro-inflammatory cytokines IL–1β, IL–6, and TNF–α using qPCR and ELISA. Blocking signal-regulated JNK–MAPK and RelA(p65) pathways significantly decreased PotD-induced pro-inflammatory cytokine production. Overall, we conclude that vaccination of PotD could induce both humoral and cellular immune responses and provide immunoprotection against H. parasuis challenge. The data also suggest that Glaesserella PotD is a novel pro-inflammatory mediator and induces TLR4-dependent pro-inflammatory activity in Raw 264.7 macrophages through JNK–MAPK and RelA(p65) pathways.

The YfeA gene, belonging to the well-conserved ABC (ATP-binding cassette) transport system Yfe, encodes the substrate-binding subunit of the iron, zinc, and manganese transport system in bacteria. As a potential vaccine candidate in Glaesserella parasuis, the functional mechanisms of YfeA in the infection process remain obscure. In this study, vaccination with YfeA effectively protected the C56BL6 mouse against the G. parasuis SC1401 challenge. Bioinformatics analysis suggests that YfeA is highly conserved in G. parasuis, and its metal-binding sites have been strictly conserved throughout evolution. Stimulation of RAW 264.7 macrophages with YfeA verified that toll-like receptors (TLR) 2 and 4 participated in the positive transcription and expression of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. The activation of TLR2 and TLR4 utilized the MyD88/MAL and TRIF/TRAM pairs to initiate TLRs signaling. Furthermore, YfeA was shown to stimulate nuclear translocation of NF-κB and activated diverse mitogen-activated protein (MAP) kinase signaling cascades, which are specific to the secretion of particular cytokine(s) in murine macrophages. Separate blocking TLR2, TLR4, MAPK, and RelA (p65) pathways significantly decreased YfeA-induced pro-inflammatory cytokine production. In addition, YfeA-stimulated RAW 264.7 produces the pro-inflammatory hallmark, reactive oxygen species (ROS). In conclusion, our findings indicate that YfeA is a novel pro-inflammatory mediator in G. parasuis and induces TLR2 and TLR4-dependent pro-inflammatory activity in RAW 264.7 macrophages through P38, JNK-MAPK, and NF-κB signaling pathways.

Pasteurella multocida, a zoonotic pathogen that produces a 146-kDa modular toxin (PMT), causes progressive atrophic rhinitis with severe turbinate bone degradation in pigs. However, its mechanism of cytotoxicity remains unclear. In this study, we expressed PMT, purified it in a prokaryotic expression system, and found that it killed PK15 cells. The host factor CXCL8 was significantly upregulated among the differentially expressed genes in a transcriptome sequencing analysis and qPCR verification. We constructed a CXCL8-knockout cell line with a CRISPR/Cas9 system and found that CXCL8 knockout significantly increased resistance to PMT-induced cell apoptosis. CXCL8 knockout impaired the cleavage efficiency of apoptosis-related proteins, including Caspase3, Caspase8, and PARP1, as demonstrated with Western blot. In conclusion, these findings establish that CXCL8 facilitates PMT-induced PK15 cell death, which involves apoptotic pathways; this observation documents that CXCL8 plays a key role in PMT-induced PK15 cell death.