EspP2 Regulates the Adhesion of Glaesserella parasuis via Rap1 Signaling Pathway.

Different levels of EspP2 expression are seen in strains of Glaesserella parasuis with high and low pathogenicity. As a potential virulence factor for G. parasuis, the pathogenic mechanism of EspP2 in infection of host cells is not clear. To begin to elucidate the effect of EspP2 on virulence, we used G. parasuis SC1401 in its wild-type form and SC1401, which was made EspP2-deficient. We demonstrated that EspP2 causes up-regulation of claudin-1 and occludin expression, thereby promoting the adhesion of G. parasuis to host cells; EspP2-deficiency resulted in significantly reduced adhesion of G. parasuis to cells. Transcriptome sequencing analysis of EspP2-treated PK15 cells revealed that the Rap1 signaling pathway is stimulated by EspP2. Blocking this pathway diminished occludin expression and adhesion. These results indicated that EspP2 regulates the adhesion of Glaesserella parasuis via Rap1 signaling pathway.

HSP90AB1 is a host factor that promotes porcine deltacoronavirus replication.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus. It causes mortality in neonatal piglets and is of growing concern because of its broad host range, including humans. To date, the mechanism of PDCoV infection remains poorly understood. Here, based on a genome-wide CRISPR screen of PDCoV-infected cells, we found that HSP90AB1 (heat shock protein 90 alpha family class B1) promotes PDCoV infection. Knockdown or KO of HSP90AB1 in LLC-PK cells resulted in a significantly suppressed PDCoV infection. Infected cells treated with HSP90 inhibitors 17-AAG and VER-82576 also showed a significantly suppressed PDCoV infection, although KW-2478, which does not affect the ATPase activity of HSP90AB1, had no effect on PDCoV infection. We found that HSP90AB1 interacts with the N, NS7, and NSP10 proteins of PDCoV. We further evaluated the interaction between N and HSP90AB1 and found that the C-tail domain of the N protein is the HSP90AB1-interacting domain. Further studies showed that HSP90AB1 protects N protein from degradation via the proteasome pathway. In summary, our results reveal a key role for HSP90AB1 in the mechanism of PDCoV infection and contribute to provide new host targets for PDCoV antiviral research.

Porcine reproductive and respiratory syndrome virus upregulates SMPDL3B to promote viral replication by modulating lipid metabolism.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a severe threat to the health of pigs globally. Host factors play a critical role in PRRSV replication. Using PRRSV as a model for genome-scale CRISPR knockout (KO) screening, we identified a host factor critical to PRRSV infection: sphingomyelin phosphodiesterase acid-like 3B (SMPDL3B). Our findings show that SMPDL3B restricted PRRSV attachment, entry, replication, and secretion and that its depletion significantly inhibited PRRSV proliferation, indicating that SMPDL3B plays a positive role in PRRSV replication. Our data also show that SMPDL3B deficiency resulted in an accumulation of intracellular lipid droplets (LDs). The expression level of key genes (ACC, SCD-1, and FASN) involved in lipogenesis was increased, whereas the fundamental lipolysis gene, ATGL, was inhibited when SMPDL3B was knocked down. Overall, our findings suggest that SMPDL3B deficiency can effectively inhibit viral infection through the modulation of lipid metabolism.

Identification of a Novel Linear B-Cell Epitope of HbpA Protein from Glaesserella parasuis Using Monoclonal Antibody.

Glaesserella parasuis (G. parasuis.) is the etiological pathogen of Glässer's disease, which causes high economic losses to the pig industry. The heme-binding protein A precursor (HbpA) was a putative virulence-associated factor proposed to be potential subunit vaccine candidate in G. parasuis. In this study, three monoclonal antibodies (mAb) 5D11, 2H81, and 4F2 against recombinant HbpA (rHbpA) of G. parasuis SH0165 (serotype 5) were generated by fusing SP2/0-Ag14 murine myeloma cells and spleen cells from BALB/c mice immunized with the rHbpA. Indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA) demonstrated that the antibody designated 5D11 showed a strong binding affinity with the HbpA protein and was chosen for subsequent experiments. The subtypes of the 5D11 were IgG1/κ chains. Western blot analysis showed that mAb 5D11 could react with all 15 serotype reference strains of G. parasuis. None of the other bacteria tested reacted with 5D11. In addition, a linear B-cell epitope recognized by 5D11 was identified by serial truncations of HbpA protein and then a series of truncated peptides were synthesized to define the minimal region that was required for mAb 5D11 binding. The 5D11 epitope was located on amino acids 324-LPQYEFNLEKAKALLA-339 by testing the 5D11 monoclonal for reactivity with 14 truncations. The minimal epitope 325-PQYEFNLEKAKALLA-339 (designated EP-5D11) was pinpointed by testing the mAb 5D11 for reactivity with a series of synthetic peptides of this region. The epitope was highly conserved among G. parasuis strains, confirmed by alignment analysis. These results indicated that mAb 5D11 and EP-5D11 might potentially be used to develop serological diagnostic tools for G. parasuis. Three-dimensional structural analysis revealed that amino acids of EP-5D11 were in close proximity and may be exposed on the surface of the HbpA protein.

The molecular diversity of transcriptional factor TfoX is a determinant in natural transformation in Glaesserella parasuis.

Natural transformation is a mechanism by which a particular bacterial species takes up foreign DNA and integrates it into its genome. The swine pathogen Glaesserella parasuis (G. parasuis) is a naturally transformable bacterium. The regulation of competence, however, is not fully understood. In this study, the natural transformability of 99 strains was investigated. Only 44% of the strains were transformable under laboratory conditions. Through a high-resolution melting curve and phylogenetic analysis, we found that genetic differences in the core regulator of natural transformation, the tfoX gene, leads to two distinct natural transformation phenotypes. In the absence of the tfoX gene, the highly transformable strain SC1401 lost its natural transformability. In addition, when the SC1401 tfoX gene was replaced by the tfoX of SH0165, which has no natural transformability, competence was also lost. These results suggest that TfoX is a core regulator of natural transformation in G. parasuis, and that differences in tfoX can be used as a molecular indicator of natural transformability. Transcriptomic and proteomic analyses of the SC1401 wildtype strain, and a tfoX gene deletion strain showed that differential gene expression and protein synthesis is mainly centered on pathways related to glucose metabolism. The results suggest that tfoX may mediate natural transformation by regulating the metabolism of carbon sources. Our study provides evidence that tfoX plays an important role in the natural transformation of G. parasuis.

A Comparative Transcriptomic Analysis Reveals That HSP90AB1 Is Involved in the Immune and Inflammatory Responses to Porcine Deltacoronavirus Infection.

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 (HSP90AB1KO) were constructed and a comparative transcriptomic analysis between PDCoV-infected HSP90AB1WT and HSP90AB1KO 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 HSP90AB1WT and HSP90AB1KO 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 HSP90AB1WT 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.

Porcine Deltacoronavirus (PDCoV) Entry into PK-15 Cells by Caveolae-Mediated Endocytosis.

(1) Background: Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus affecting pig breeding industries worldwide, and its pathogenic mechanism remains unclear. (2) Methods: In this study, we preliminarily identified the endocytic pathway of PDCoV in PK-15 cells, using six chemical inhibitors (targeting clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis pathway and endosomal acidification), overexpression of dominant-negative (DN) mutants to treat PK-15 cells and proteins knockdown. (3) Results: The results revealed that PDCoV entry was not affected after treatment with chlorpromazine (CPZ), 5-(N-ethyl-N-isopropyl) amiloride (EIPA)or ammonium chloride (NH4Cl), indicating that the entry of PDCoV into PK-15 cells were clathrin-, micropinocytosis-, PH-independent endocytosis. Conversely, PDCoV infection was sensitive to nystatin, dynasore and methyl-ß-cyclodextrin (MßCD) with reduced PDCoV internalization, indicating that entry of PDCoV into PK-15 cells was caveolae-mediated endocytosis that required dynamin and cholesterol; indirect immunofluorescence and shRNA interference further validated these results. (4) Conclusions: In conclusion, PDCoV entry into PK-15 cells depends on caveolae-mediated endocytosis, which requires cholesterol and dynamin. Our finding is the first initial identification of the endocytic pathway of PDCoV in PK-15 cells, providing a theoretical basis for an in-depth understanding of the pathogenic mechanism of PDCoV and the design of new antiviral targets.

Study of the inhibitory effect of STAT1 on PDCoV infection.

Porcine deltacoronavirus (PDCoV) is an enteropathogen found in many pig producing countries. It can cause acute diarrhea, vomiting, dehydration, and death in newborn piglets, seriously affecting the development of pig breeding industries. To date, our knowledge of the pathogenesis of PDCoV and its interactions with host cell factors remains incomplete. Using Co-IP coupled with LC/MS-MS, we identified 67 proteins that potentially interact with PDCoV in LLC-PK1 cells; five of the identified proteins were chosen for further evaluation (IMMT, STAT1, XPO5, PIK3AP1, and TMPRSS11E). Five LLC-PK1 cell lines, each with one of the genes of interest knocked down, were constructed using CRISPR/cas9. In these knockdown cells lines, only STAT1KD resulted in a significantly greater virus yield. Knockdown of the remaining four genes resulted, to varying degrees, in a lower virus yield that wild-type LLC-PK1 cells. The absence of STAT1 did not significantly affect the attachment of PDCoV to cells, but did result in increased viral internalization. Additionally, PDCoV infection stimulated expression of interferon stimulated genes (ISGs) downstream of STAT1 (IFIT1, IFIT2, RADS2, ISG15, MX1, and OAS1) while knockdown of STAT1 resulted in a greater than 80 % decrease in the expression of all six ISGs. Our findings show that STAT1 interacts with PDCoV, and plays a negative regulatory role in PDCoV infection.

Development and application of a visual microarray for synchronously detecting H5N1, H7N9 and H9N2 avian influenza virus RNA.

The aim of this study was to develop a microarray assay for the simultaneous detection of the H5, H7, H9, N1, N9 and N2 genes of the avian influenza virus (AIV) using a Nanogold-streptavidin and silver-stain-enhanced nucleic acid dot-blot hybridisation system. The conserved sequences of H5 genes from H5N1, H7 genes from H7N9, H9 genes from H9N2, N9 genes from H7N9 and N2 genes from H9N2 AIV were cloned, together with that of N1 obtained commercially, and were used as templates for generating the probes using biotin-labeled primers, which targeted the conserved regions of H5, H7, H9, N1, N9 and N2 genes, respectively. The oligonucleotide probes were diluted using the spotting buffer and ddH2O, and each probe was then spotted to each specific position on the microarray. The PCR products including biotin-labeled lambda, NP, H5, H7, H9, N1, N9 and N2 were mixed, 200 µL of which was then added to the microarray chamber after denaturing. Following a hybridization incubation at 45℃ for 120 min, the microarray was then incubated with nanogold-streptavidin about 4 µg/mL for 30 min. After the supplementary of 200 µL of silver buffer A and silver buffer B in the chamber, the hybridization results were assessed by direct visualization in the dark at room temperature. The microarray assay was optimized and its specificity, sensitivity and stability were evaluated. The optimal conditions comprised a probe concentration of 50 µmol/L, a hybridization temperature of 45℃ and a hybridization time of 2 h. The optimal concentration of nanogold-streptavidin was 4 µg/mL and the optimal staining time was 7 min. The results of specificity evaluation showed that no cross-binding of the probes with each other and no cross-hybridization with Newcastle disease virus, infectious bronchitis virus and infectious laryngotracheitis virus was observed. The optimized microarray assay was significantly more sensitivity than the reverse-transcription PCR assay. The microarray was available after storing at less 90 d at 4 ℃. The optimized microarray assay was validated on clinical specimens and the results showed that it had over 95.6 % correlation with reverse-transcription PCR method. Therefore, the microarray assay could be used for the high throughput detection of AIV infections due to H5N1, H7N9 and H9N2.

HSP90 inhibitors 17-AAG and VER-82576 inhibit porcine deltacoronavirus replication in vitro.

Porcine deltacoronavirus (PDCoV) is highly pathogenic to piglets, and no specific drugs or vaccines are available for the prevention and treatment of PDCoV infection, the need for antiviral therapies is pressing. HSP90 inhibitors have potent inhibitory effects against the replication of numerous viruses, hence we evaluated three HSP90 inhibitors, 17-AAG, VER-82576, and KW-2478, for their effects on PDCoV infection in vitro. We evaluated their effectivenesses at suppressing PDCoV by qRT-PCR, western blot, and TCID50 assay, and found that 17-AAG and VER-82576 inhibited PDCoV at the early stage of replication, while KW-2478 showed no significant antiviral activity at any stage of infection. These results indicated that the PDCoV-inhibitory effects of 17-AAG and VER-82576 might be exerted by targeting host cell factor HSP90AB1 but not HSP90AA1. Further study showed that HSP90AB1 mRNA and protein levels were not significantly different in 17-AAG and VER-82576-treated cells versus control cells. 17-AAG and VER-82576 were also evaluated for their effects on the expressions of TNF-α, IL-6, and IL-12, which are PDCoV-induced proinflammatory cytokines. We found that both 17-AAG and VER-82576 inhibited the expressions of TNF-α, IL-6, and IL-12 to varying degrees, but in a dose dependent manner. From our data we can conclude that the HSP90 inhibitors 17-AAG and VER-82576 are promising candidates for the treatment of PDCoV infection.

Comparative transcriptome analysis reveals that deletion of CheY influences gene expressions of ABC transports and metabolism in Haemophilus parasuis.

Haemophilus (Glaesserella) parasuis is a commensal bacterium that causes Glässer's disease (GD) in swine. As a global transcriptional factor, CheY regulates the expression of hundreds of genes in H. parasuis. In this study, we measured changes in gene expression at the whole transcriptome level using RNAseq. We identified 2058 co-expressed genes, and found 624 differentially expressed genes (q < 0.05) in ΔcheY and SC1401. Several important GO annotations and signaling pathways were identified. RNA-seq results were assembled according to the reference genome, compared with the annotated gene model, and 12 new transcriptional regions were found. Finally, q-PCR results validated the RNA-seq results with 8 randomly selected genes. The present study indicated that CheY is mainly involved in the regulation of ABC transport, oxidative phosphorylation, and ß-Lactam resistance. We draw the regulatory network of CheY, which offers greater insight into the regulatory mechanism of CheY in H.parasuis.

Deletion of Polyamine Transport Protein PotD Exacerbates Virulence in Glaesserella (Haemophilus) parasuis in the Form of Non-biofilm-generated Bacteria in a Murine Acute Infection Model.

Polyamines are small, polycationic molecules with a hydrocarbon backbone and multiple amino groups required for optimal cell growth. The potD gene, belonging to the ABC (ATP-binding cassette) transport system potABCD, encodes the bacterial substrate-binding subunit of the polyamine transport system, playing a pivotal role in bacterial metabolism and growth. The swine pathogen Glaesserella parasuis possesses an intact pot operon, and the studies presented here mainly examined the involvement of PotD in Glaesserella pathogenesis. A potD-deficient mutant was constructed using a virulent G. parasuis strain SC1401 by natural transformation; immuno-electron microscopy was used to identify the subcellular location of native PotD protein; an electron microscope was adopted to inspect biofilm and bacterial morphology; immunofluorescence technique was employed to study cellular adhesion, the levels of inflammation and apoptosis. The TSA++-pre-cultured mutant strain showed a significantly reduced adhesion capacity to PK-15 and MLE-12 cells. Likewise, we also found attenuation in virulence using murine models focusing on the clinical sign, H&E, and IFA for inflammation and apoptosis. However, when the mutant was grown in TSB++, virulence recovered to normal levels, along with a high level of radical oxygen species formation in the host. The expression of PotD could actively stimulate the production of ROS in Raw 264.7. Our data suggested that PotD from G. parasuis has a high binding potential to polyamine, and is essential for the full bacterial virulence within mouse models. However, the virulence of the potD mutant is highly dependent on its TSA++ culture conditions rather than on biofilm-formation.

Identification of a Novel Linear B-Cell Epitope on the Nucleocapsid Protein of Porcine Deltacoronavirus.

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.

Identification of the immunodominant neutralizing regions in the spike glycoprotein of porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV), is an emerging enteropathogenic coronavirus in pigs, that poses a novel threat to swine husbandry worldwide. Crucial to halting PDCoV transmission and infection is the development of effective therapies and vaccines. The spike (S) protein of coronavirus is the major target of host neutralizing antibodies, however the immunodominant neutralizing region in the S protein of PDCoV has not been defined. Here, three truncations of the PDCoV S protein were generated, the N-terminal domain of the S1 subunit (NTD, amino acids (aa) 50-286), the C-terminal domain of the S1 subunit (CTD, aa 278-616), and S2 subunit (aa 601-1087). The proteins were expressed using an E. coli expression system. Polyclonal antisera against the three recombinant proteins were produced in rabbits and mice. All three antisera were able to inhibit PDCoV infection in vitro, as determined by virus neutralization assay, fluorescent focus neutralization assay, and plaque-reduction neutralization. The CTD-specific antisera had the most potent PDCoV-neutralizing effect, indicating that the CTD region may contain the major neutralizing epitope(s) in the PDCoV S protein. Based on these findings, CTD may be a promising target for development of an effective vaccine against PDCoV infection in pigs.

Polyamine Transport Protein PotD Protects Mice against Haemophilus parasuis and Elevates the Secretion of Pro-Inflammatory Cytokines of Macrophage via JNK-MAPK and NF-κB Signal Pathways through TLR4.

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-1ß, 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.

Hsp40 Protein DNAJB6 Interacts with Viral NS3 and Inhibits the Replication of the Japanese Encephalitis Virus.

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.

Characterization and Pathogenicity of the Porcine Deltacoronavirus Isolated in Southwest China.

Porcine deltacoronavirus (PDCoV) is a newly emerging enteric pathogen in swine that causes diarrhea in neonatal piglets and creates an additional economic burden on porcine industries in Asia and North America. In this study, a PDCoV isolate, CHN-SC2015, was isolated from Sichuan Province in southwest China. The isolate was characterized by a cytopathic effect, immunofluorescence, and electron microscopy. CHN-SC2015 titers in LLC-PK cells ranged from 104.31 to 108.22 TCID50/mL during the first 30 passages. During serial passage, 11 nucleotide mutations occurred in the S gene, resulting in nine amino acid changes. A whole genome sequencing analysis demonstrated that CHN-SC2015 shares 97.5%-99.1% identity with 59 reference strains in GenBank. Furthermore, CHN-SC2015 contained 6-nt deletion and 9-nt insertion in the ORF1ab gene, 3-nt deletion in the S gene and 11-nt deletion in its 3'UTR compared with other reference strains available in GenBank. A phylogenetic analysis showed that CHN-SC2015 is more closely related to other PDCoV strains in China than to the strains from Southeast Asia, USA, Japan, and South Korea, indicating the diversity of genetic relationships and regional and epidemic characteristics among these strains. A recombination analysis indicated that CHN-SC2015 experienced recombination events between SHJS/SL/2016 and TT-1115. In vivo infection demonstrated that CHN-SC2015 is highly pathogenic to sucking piglets, causing diarrhea, vomiting, dehydration, and death. Virus was shed daily in the feces of infected piglets and upon necropsy, was found distributed in the gastrointestinal tract and in multiple organs. CHN-SC2015 is the first systematically characterized strain from southwest China hitherto reported. Our results enrich the body of information on the epidemiology, pathogenicity and molecular evolution associated with PDCoV.

Enhanced Immune Responses Against Japanese Encephalitis Virus Infection Using Japanese Encephalitis Live-Attenuated Virus Adjuvanted with Montanide GEL 01 ST in Mice.

Japanese encephalitis virus (JEV) is one of the major causes of acute encephalitis in human and animal. To prevent JEV infection, an effective live-attenuated vaccine is needed. In the article, JEV attenuated strain, SCYA201201 of GI genotype, which was mixed with 10% concentrate GEL 01 ST adjuvant (Montanide™ GEL 01 ST), was selected for a vaccine candidate and its immunogenicity was evaluated in mice. Our results showed that JEV mixed with GEL 01 ST elicited production of both IgG1 and IgG2a antibodies, and enhanced virus-specific crossprotective intergenotypic response in mice. Proliferation of splenocytes was observed in all immunized groups and a relatively higher proliferation activity was detected in JEV mixed with GEL 01 ST group (p < 0.05). In the JEV + 10% GEL 01 ST vaccinated group, the proportion of CD4+ T cells in spleen was significantly higher than that of control group (p < 0.05), and the yields of interleukin (IL)-2, IL-4, and interferon-γ in the splenocyte supernatant were also significantly higher than that of control group (p < 0.05). Moreover, complete protection was provided after JEV challenge in mice in JEV mixed with GEL 01 ST group, and early immunity was detected in those mice immunized with JEV mixed with GEL 01 ST. These findings confirm that GEL 01 ST can enhance JEV live-attenuated immunogenicity, and JEV +10% GEL 01 ST used as vaccine candidates provide protection against JEV infection in a mouse model, which could be used as potential vaccine candidates in pig.

A requirement of TolC1 for effective survival, colonization and pathogenicity of Actinobacillus pleuropneumoniae.

To establish infection in the host, pathogens have evolved sophisticated systems to cope with environmental conditions and to protect cells against host immunity. TolC is the outer membrane channel component of type 1 secretion systems and multidrug efflux pumps that plays critical roles during the infection process in many pathogens. However, little is known about the exact roles of TolC1 in the pathogenicity of A. pleuropneumoniae, an etiological agent of the porcine contagious pleuropneumoniae that causes severe respiratory disease. In this study, deletion of tolC1 causes apparent ultrastructural defects in A. pleuropneumoniae cell examined by transmission electron microscopy. The tolC1 mutant is hypersensitivity to oxidative, osmotic and acid challenges by in vitro stress assays. Analysis on secreted proteins shows that the excretion of ApxIIA and an ApxIVA-like protein, ApxIVA-S, is abolished in the absence of TolC1. This result confirms the essential role of TolC1 in the secretion of Apx toxins and this is the first identification of an ApxIVA-like protein in in vitro culture of A. pleuropneumoniae. Besides, disruption of TolC1 leads to a significant attenuation of virulence in mice by an intraperitoneal route of A. pleuropneumoniae. The basis for the attenuation is further investigated using a mouse intranasal infection model, which reveals an impaired ability to colonize and induce lesions in the lungs for the loss of TolC1 of A. pleuropneumoniae. In conclusion, our findings demonstrate significant roles of TolC1 in facilitating bacterial survival in hostile conditions, maximum colonization as well as pathogenicity during the infection of A. pleuropneumoniae.

Construction of targeted and integrative promoter-reporter plasmids pDK-K and pDK-G to measure gene expression activity in Haemophilus parasuis.

Haemophilus parasuis (H. parasuis) is rather difficult to manipulate genetically due to the diversity of restriction-modification systems and other mechanisms harbored by various isolates. This prevents exogenous plasmids from replicating in this species and hinders research efforts focused on transcriptional regulators in this bacterium. In this study, we generated a convenient promoter reporter system based on gene knock-in method using natural transformation in H. parasuis. Gene knock-in has proven useful as a powerful tool facilitating identification and studying the transcription activities of regulators under a variety of conditions that favor gene transcription or expression from an incorporated promoter. The vectors, pDK-K and pDK-G, carrying promoterless reporter lacZ gene and two homologous sequences flanking a knock-in site, may have some advantages over the extensively used plasmid-bearing reporter system in other bacteria in stability and ease of genetic manipulation in H. parasuis. The knock-in site was positioned at a site occupied by flanking genes that were both hypothetical and had the same transcription orientation, thus the expression of the reversely cloned promoter-lacZ fusion wouldn't be affected by the upstream promoter on the chromosome. The expression activity of lacZ gene under the transcriptional activation of a 300 bp promoter-proximal segment of cyaA, crp or comA genes in H. parasuis was separately validated using X-gal and o-nitrophenyl-ß-d-galactoside(ONPG) as substrates. The derivatives harboring promoter-lacZ fusion segments showed significantly higher ß-galactosidase activity levels than the promoterlessones both in TSB++ broth and on TSA++ plate as screened either by X-gal method or the standard Miller method. We also used pDK vector to further certify that the cyaA promoter is inducible and whose transcriptional levels were in correlation with the growth kinetics of the bacteria in TSB++. With this system, gene knock-in method based on natural transformation in H. parasuis proved to be useful in identifying transcriptional regulation of a certain promoter.