Identification of abnormal closed-loop pathways in patients with MRI-negative pharmacoresistant epilepsy.

Epilepsy is a disorder of brain networks, that is usually combined with cognitive and emotional impairment. However, most of the current research on closed-loop pathways in epilepsy is limited to the neuronal level or has focused only on known closed-loop pathways, and studies on abnormalities in closed-loop pathways in epilepsy at the whole-brain network level are lacking. A total of 26 patients with magnetic resonance imaging-negative pharmacoresistant epilepsy (MRIneg-PRE) and 26 healthy controls (HCs) were included in this study. Causal brain networks and temporal-lag brain networks were constructed from resting-state functional MRI data, and the Johnson algorithm was used to identify stable closed-loop pathways. Abnormal closed-loop pathways in the MRIneg-PRE cohort compared with the HC group were identified, and the associations of these pathways with indicators of cognitive and emotional impairments were examined via Pearson correlation analysis. The results revealed that the abnormal stable closed-loop pathways were distributed across the frontal, parietal, and occipital lobes and included altered functional connectivity values both within and between cerebral hemispheres. Four abnormal closed-loop pathways in the occipital lobe were associated with emotional and cognitive impairments. These abnormal pathways may serve as biomarkers for the diagnosis and guidance of individualized treatments for MRIneg-PRE patients.

Regulating pathway for bacterial diversities toward improved ecological benefits of thiencarbazone-methyl·isoxaflutole application: A field experiment.

Herbicide abuse has a significantly negative impact on soil microflora and further influences the ecological benefit. The regulating measures and corresponding mechanisms mitigating the decreased bacterial diversity due to herbicide use have rarely been studied. A field experiment containing the application gradient of an efficient maize herbicide thiencarbazone-methyl·isoxaflutole was performed. The relationship between soil bacterial community and thiencarbazone-methyl·isoxaflutole use was revealed. Modified attapulgite was added to explore its impacts on soil microflora under the thiencarbazone-methyl·isoxaflutole application. Based on the analytic network process-entropy weighting method-TOPSIS method model, the ecological benefit focusing on microbial responses was quantitatively estimated along with technical effectiveness and economic benefit. The results showed that the diversity indices of soil microflora, especially the Inv_Simpson index, were reduced at the recommended, 5 and 10 times the recommended dosages of thiencarbazone-methyl·isoxaflutole use. The Flavisolibacter bacteria was negatively correlated with the residues in soils based on the random forest model and correlation analysis, indicating a potential degrader of thiencarbazone-methyl·isoxaflutole residues. The structural equation model further confirmed that the high soil water content and soil pH promoted the function of Flavisolibacter bacteria, facilitated the dissipation of thiencarbazone-methyl·isoxaflutole residues and further improved the diversity of soil microflora. In addition, the presence of modified attapulgite was found to increase the soil pH, which may improve bacterial diversity through the regulating pathway. This explained the high ecological benefits of the treatment where the thiencarbazone-methyl·isoxaflutole was applied at the recommended dosage rates in conjunction with modified attapulgite addition. Therefore, the comprehensive benefits of thiencarbazone-methyl·isoxaflutole application with a focus on ecological benefits can be improved by regulating the soil pH with modified attapulgite.

Evaluation of the immunoprotective effects of porcine deltacoronavirus subunit vaccines.

Porcine deltacoronavirus (PDCoV), a new porcine enteric coronavirus, has seriously endangered the pig breeding industry and caused great economic losses. However, a PDCoV vaccine is not commercially available. Therefore, new and efficient PDCoV vaccines must be developed without delay. In this study, we used the ExpiCHO eukaryotic expression system to express and purify the following 3 structural proteins of PDCoV: S, N and M. Subsequently, the level of humoral and cellular immunity induced by the S protein (immunization with the S protein alone) and a protein mixture (immunization with a mixture of S, N and M proteins) were evaluated in mice and piglets, respectively, and the performances of the 2 immunizations in a challenge protection test were assessed in piglets. The results showed that both the S protein and the protein mixture induced the production of high levels of specific IgG antibodies and neutralizing antibodies and effectively neutralized PDCoV-infected LLC-PK cells in vitro. Furthermore, compared with the S protein, the N and M proteins in the protein mixture promoted the expression of CD8+ T cells and IFN-γ, induced a stronger cellular immune response, and effectively protected 4/5 of the piglets from PDCoV infection. In conclusion, the results of this study showed that the N and M proteins play important roles in inducing an immunoprotective response. Using N and M antigens as effective antigenic components in the development of PDCoV vaccines in the future will effectively increase the immune efficacy of the vaccines.

High definition transcranial direct current stimulation of the Cz improves social dysfunction in children with autism spectrum disorder: A randomized, sham, controlled study.

The purpose of this study was to determine the effect of the Cz of high-definition 5-channel tDCS (HD-tDCS) on social function in 4-12 years-old children with autism spectrum disorder (ASD). This study was a randomized, double-blind, pseudo-controlled trial in which 45 ASD children were recruited and divided into three groups with sex, age, and rehabilitation treatment as control variables. Each group of 15 children with ASD was randomly administered active HD-tDCS with the Cz as the central anode, active HD-tDCS with the left dorsolateral prefrontal cortex (F3) as the central anode, and sham HD-tDCS with the Cz as the central anode with 14 daily sessions in 3 weeks. The Social Responsiveness Scale Chinese Version (SRS-Chinese Version) was compared 1 week after stimulation with values recorded 1 week prior to stimulation. At the end of treatment, both the anodal Cz and anodal left DLFPC tDCS decreased the measures of SRS-Chinese Version. The total score of SRS-Chinese Version decreased by 13.08%, social cognition decreased by 18.33%, and social communication decreased by 10.79%, which were significantly improved over the Cz central anode active stimulation group, especially in children with young age, and middle and low function. There was no significant change in the total score and subscale score of SRS-Chinese Version over the Cz central anode sham stimulation group. In the F3 central anode active stimulation group, the total score of SRS-Chinese Version decreased by 13%, autistic behavior decreased by 19.39%, and social communication decreased by 14.39%, which were all significantly improved. However, there was no significant difference in effect between the Cz and left DLPFC stimulation conditions. HD-tDCS of the Cz central anode may be an effective treatment for social dysfunction in children with ASD.

Adenovirus-vectored PDCoV vaccines induce potent humoral and cellular immune responses in mice.

Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes severe watery diarrhea, vomiting, dehydration and high mortality in piglets, resulting in significant economic losses by the global pig industry. Recently, PDCoV has also shown the potential for cross-species transmission. However, there are currently few vaccine studies and no commercially available vaccines for PDCoV. Hence, here, two novel human adenovirus 5 (Ad5)-vectored vaccines expressing codon-optimized forms of the PDCoV spike (S) glycoprotein (Ad-PD-tPA-Sopt) and S1 glycoprotein (Ad-PD-oriSIP-S1opt) were constructed, and their effects were evaluated via intramuscular (IM) injection in BALB/c mice with different doses and times. Both vaccines elicited robust humoral and cellular immune responses; moreover, Ad-PD-tPA-Sopt-vaccinated mice after two IM injections with 108 infectious units (IFU)/mouse had significantly higher anti-PDCoV-specific neutralizing antibody titers. In contrast, the mice immunized with Ad-PD-tPA-Sopt via oral gavage (OG) did not generate robust systemic and mucosal immunity. Thus, IM Ad-PD-tPA-Sopt administration is a promising strategy against PDCoV and provides useful information for future animal vaccine development.

High-Efficiency and Reliable Value Geometric Standard: Integrated Periodic Structure Reference Materials.

Integrated periodic structure reference materials are crucial for calibration in optical instruments and micro-computed tomography (micro-CT), yet they face limitations concerning a restricted measurement range, a single pattern type, and a single calibration parameter. In this study, we address these challenges by developing integrated periodic structure reference materials with an expanded measurement range, diverse pattern types, and multiple calibration parameters through a combination of photolithography and inductively coupled plasma (ICP) etching process. These reference materials facilitate high-efficiency and multi-value calibration, finding applications in the calibration of optical instruments and micro-CT systems. The simulations were conducted using MATLAB (R2022b) to examine the structure-morphology changes during the single-step ICP etching process. The variation rules governing line widths, periods, etching depths, and side wall verticality in integrated periodic structure reference materials were thoroughly evaluated. Linewidths were accurately extracted utilizing an advanced image processing algorithm, while average period values were determined through the precise Fast Fourier Transform method. The experimental results demonstrate that the relative errors of line widths do not exceed 17.5%, and the relative errors of periods do not exceed 1.5%. Furthermore, precise control of the etching depth was achieved, ranging from 30 to 60 µm for grids with line widths 2-20 µm. The side wall verticality exhibited remarkable consistency with an angle of 90° ± 0.8°, and its relative error was found to be less than 0.9%.

Nicotinamide Efficiently Suppresses Porcine Epidemic Diarrhea Virus and Porcine Deltacoronavirus Replication.

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV), members of the genus Coronavirus, mainly cause acute diarrhea, vomiting and dehydration in piglets, and thus lead to serious economic losses. In this study, we investigated the effects of nicotinamide (NAM) on PEDV and PDCoV replication and found that NAM treatment significantly inhibited PEDV and PDCoV reproduction. Moreover, NAM plays an important role in replication processes. NAM primarily inhibited PEDV and PDCoV RNA and protein synthesis rather than other processes. Furthermore, we discovered that NAM treatment likely inhibits the replication of PEDV and PDCoV by downregulating the expression of transcription factors through activation of the ERK1/2/MAPK pathway. Overall, this study is the first to suggest that NAM might be not only an important antiviral factor for swine intestinal coronavirus, but also a potential candidate to be evaluated in the context of other human and animal coronaviruses.

Recombinant human adenovirus type 5 based vaccine candidates against GIIa- and GIIb-genotype porcine epidemic diarrhea virus induce robust humoral and cellular response in mice.

Porcine epidemic diarrhea virus (PEDV) is a porcine enteropathogenic coronavirus causing severe watery diarrhea, vomiting, dehydration, and death in piglets. However, most commercial vaccines are developed based on the GI genotype strains, and have poor immune protection against the currently dominant GII genotype strains. Therefore, four novel replication-deficient human adenovirus 5-vectored vaccines expressing codon-optimized forms of the GIIa and GIIb strain spike and S1 glycoproteins were constructed, and their immunogenicity was evaluated in mice by intramuscular (IM) injection. All the recombinant adenoviruses generated robust immune responses, and the immunogenicity of recombinant adenoviruses against the GIIa strain was stronger than that of recombinant adenoviruses against the GIIb strain. Moreover, Ad-XT-tPA-Sopt-vaccinated mice elicited optimal immune effects. In contrast, mice immunized with Ad-XT-tPA-Sopt by oral gavage did not induce strong immune responses. Overall, IM administration of Ad-XT-tPA-Sopt is a promising strategy against PEDV, and this study provides useful information for developing viral vector-based vaccines.

Identification of B-cell epitopes on structural proteins VP1 and VP2 of Senecavirus A and development of a multi-epitope recombinant protein vaccine.

Senecavirus A (SVA) is an important pathogenic cause of vesicular disease in pigs worldwide. In this study, we screened the B-cell epitopes of SVA using a bioinformatics approach combined with an overlapping synthetic polypeptide method. Four dominant B-cell epitopes (at amino acid (aa) positions: 7-26, 48-74, 92-109, and 129-144) from the VP1 protein and five dominant B-cell epitopes (aa: 38-57, 145-160, 154-172, 193-208, 249-284) from the VP2 protein were identified. Multi-epitope genes comprising the identified B-cell epitope domains were synthesized, prokaryotic expressed, and purified, and their immune protection efficacy was evaluated in piglets. Our results showed that the multi-epitope recombinant protein rP2 induced higher neutralizing antibodies and provided 80% protection against homologous SVA challenge. Thus, the B-cell epitope peptides identified in this study are potential candidates for SVA vaccine development, and rP2 may offer safety and efficacy in controlling infectious SVA.

Differences in the pathogenicity of Chinese virulent genotype GIIa and GIIb porcine epidemic diarrhea virus strains and the humoral immune status of one- and two-month-old weaned pigs infected with these strains.

We evaluated differences in the pathology and humoral immune status in one- and two-month-old weaned pigs infected with virulent Chinese genotype GIIa and GIIb strains of porcine epidemic diarrhea virus (PEDV). All pigs infected with the GIIa strain developed severe diarrhea (100%), while the morbidity of the GIIb strain in one- and two-month-old weaned pigs was 80% (4/5) and 40% (2/5), respectively. There was no significant difference in IgA, IgG, or virus-neutralizing (VN) antibody levels associated with GIIa and GIIb in one-month-old weaned pigs (P > 0.05), but in two-month-old weaned pigs, the IgA, IgG, and VN antibody levels associated with GIIa were significantly higher than those associated with GIIb (P < 0.05).

Social Brain Network of Children with Autism Spectrum Disorder: Characterization of Functional Connectivity and Potential Association with Stereotyped Behavior.

Objective: To identify patterns of social dysfunction in adolescents with autism spectrum disorder (ASD), study the potential linkage between social brain networks and stereotyped behavior, and further explore potential targets of non-invasive nerve stimulation to improve social disorders. Methods: Voxel-wise and ROI-wise analysis methods were adopted to explore abnormalities in the functional activity of social-related regions of the brain. Then, we analyzed the relationships between clinical variables and the statistical indicators of social-related brain regions. Results: Compared with the typically developing group, the functional connectivity strength of social-related brain regions with the precentral gyrus, postcentral gyrus, supplementary motor area, paracentral lobule, median cingulum, and paracingulum gyri was significantly weakened in the ASD group (all p < 0. 01). The functional connectivity was negatively correlated with communication, social interaction, communication + social interaction, and the total score of the ADOS scale (r = -0.38, -0.39, -0.40, and -0.3, respectively; all p < 0.01), with social awareness, social cognition, social communication, social motivation, autistic mannerisms, and the total score of the SRS scale (r = -0.32, -0.32, -0.40, -0.30, -0.28, and -0.27, respectively; all p < 0.01), and with the total score of SCQ (r = -0.27, p < 0.01). In addition, significant intergroup differences in clustering coefficients and betweenness centrality were seen across multiple brain regions in the ASD group. Conclusions: The functional connectivity between social-related brain regions and many other brain regions was significantly weakened compared to the typically developing group, and it was negatively correlated with social disorders. Social network dysfunction seems to be related to stereotyped behavior. Therefore, these social-related brain regions may be taken as potential stimulation targets of non-invasive nerve stimulation to improve social dysfunction in children with ASD in the future.

Monolithically integrated triaxial high-performance micro accelerometers with position-independent pure axial stressed piezoresistive beams.

With the increasing demand for multidirectional vibration measurements, traditional triaxial accelerometers cannot achieve vibration measurements with high sensitivity, high natural frequency, and low cross-sensitivity simultaneously. Moreover, for piezoresistive accelerometers, achieving pure axial deformation of the piezoresistive beam can greatly improve performance, but it requires the piezoresistive beam to be located in a specific position, which inevitably makes the design more complex and limits the performance improvement. Here, a monolithically integrated triaxial high-performance accelerometer with pure axial stress piezoresistive beams was designed, fabricated, and tested. By controlling synchronous displacements at both piezoresistive beam ends, the pure axial stress states of the piezoresistive beams could be easily achieved with position independence without tedious calculations. The measurement unit for the z-axis acceleration was innovatively designed as an interlocking proof mass structure to ensure a full Wheatstone bridge for sensitivity improvement. The pure axial stress state of the piezoresistive beams and low cross-sensitivity of all three units were verified by the finite element method (FEM). The triaxial accelerometer was fabricated and tested. Results showing extremely high sensitivities (x axis: 2.43 mV/g/5 V; y axis: 2.44 mv/g/5 V; z axis: 2.41 mV/g/5 V (without amplification by signal conditioning circuit)) and high natural frequencies (x/y axes: 11.4 kHz; z-axis: 13.2 kHz) were obtained. The approach of this paper makes it simple to design and obtain high-performance piezoresistive accelerometers.

Mucosal immune responses induced by oral administration of recombinant Lactococcus lactis expressing the S1 protein of PDCoV.

Porcine deltacoronavirus is an evolving coronavirus that primarily infects the intestine and may lead to intestinal disease in piglets. Up to now, no commercial vaccination is readily accessible to protect against the spread of PDCoV. Lactococcus lactis has been shown to have good immune efficacy and safety and can be used as a genetically engineered vaccine to deliver antigens. In this research, we utilized L. lactis NZ9000 to provide the S1 protein orally and improved the delivery efficiency by connecting the M cell targeting ligand Co1 with the S1 protein of PDCoV in tandem to obtain the recombinant protein S1-Co1. We successfully constructed two recombinant strains capable of expressing PDCoV-S1 and PDCoV-S1-Co1 proteins (i.e., L. lactis NZ9000-S1 and L. lactis NZ9000-S1-Co1), and their immunogenic capacity was evaluated in mice. Our study shows that Lactococcus is an advantageous bacterial live vector vaccine and is anticipated as a potential PDCoV vaccination option.

Polydopamine-coated thalidomide nanocrystals promote DSS-induced murine colitis recovery through Macrophage M2 polarization together with the synergistic anti-inflammatory and anti-angiogenic effects.

High levels of proinflammatory cytokines, macrophage polarization status and immune-mediated angiogenesis play pivotal roles in the pathogenesis of inflammatory bowel disease (IBD). Thalidomide, an anti-inflammatory, immunomodulatory and antiangiogenic agent, is used off-label for treatment of IBD. The therapeutic potential of thalidomide is limited by its poor solubility and side effects associated with its systemic exposure. To address these issues and promote its therapeutic effects on IBD, thalidomide nanocrystals (Thali NCs) were prepared and coated with polydopamine (PDA), a potential macrophage polarization modulator, to form PDA coated Thali NCs (Thali@PDA). Thali@PDA possessed a high drug loading and displayed average particle size of 764.7 ± 50.30 nm. It showed a better anti-colitis effect than bare thalidomide nanocrystals at the same dose of thalidomide. Synergistic effects of polydopamine on anti-inflammatory and anti-angiogenic activities of thalidomide were observed. Furthermore, PDA coating could direct polarization of macrophages towards M2 phenotype, which boosted therapeutic effects of Thali@PDA on IBD. Upon repeated dosing of Thali@PDA for one week, symptoms of IBD in mice were significantly relieved, and histomorphology of the colitis colons were normalized. Key proinflammatory cytokine levels in the inflamed intestines were significantly decreased. Toxicity study also revealed that Thali@PDA is a safe formulation.

Diagnostic strategy for the combined effects of microplastics and potentially toxic elements on microbial communities in catchment scale.

Combined effects of potentially toxic materials (PTMs) released from production activities on microbial communities in environmental flimsy area are poorly recognised. Microplastics (MPs) and potentially toxic elements (PTEs) were investigated in soils and river sediments in a headwater catchment from the Qinghai-Tibet Plateau. Their co-effects on microbial communities and the controlling factors affecting communities were further explored. Results showed that MPs and PTEs significantly accumulated in soils and sediments. Among which fragment-shaped MPs and copper (Cu) dominated, with mean contents of 1.11 × 104 and 1.81 × 104 items kg-1 and 13.80 and 7.33 mg kg-1 in soils and sediments, respectively. Distribution index (0.54) suggested that fiber-shaped MPs preferred to transport into rivers and deposited in sediments. The film mulching contributed significantly to the occurrence of fragment-shaped MPs, while Cu may be derived from industrial wastewater. The antagonistic effect between fiber-shaped MPs and zinc (Zn) on soil microbial structure was found based on their obtuse angle in canonical correlation analysis. While the synergistic effect between total phosphorus (TP) and Cu on diversity was detected by interaction detector model (q(TP ∩ Cu) >q(TP) >q(Cu), p < 0.05). Soil TP and Cu were identified as controlling factors influencing diversity through random forest model and factor detector (q(TP) = 0.49, q(Cu) = 0.36, p < 0.05), which may be related to direct nutrient supply and microbial resistance, respectively. The negative effects of MPs on structure might be counteracted by increasing Zn content, while the co-existence of TP and Cu further increased diversity. A diagnostic framework, which involves background data collection, sampling analysis, characterisation and relationship investigation, was proposed to explore the co-effects of complex pollution and factors on communities. This study may provide strategies to mitigate the negative effects on microorganisms in the environment.

Transcriptome Analysis of LLC-PK Cells Single or Coinfected with Porcine Epidemic Diarrhea Virus and Porcine Deltacoronavirus.

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) are the two most prevalent swine enteric coronaviruses worldwide. They commonly cause natural coinfections, which worsen as the disease progresses and cause increased mortality in piglets. To better understand the transcriptomic changes after PEDV and PDCoV coinfection, we compared LLC porcine kidney (LLC-PK) cells infected with PEDV and/or PDCoV and evaluated the differential expression of genes by transcriptomic analysis and real-time qPCR. The antiviral efficacy of interferon-stimulated gene 20 (ISG20) against PDCoV and PEDV infections was also assessed. Differentially expressed genes (DEGs) were detected in PEDV-, PDCoV-, and PEDV + PDCoV-infected cells at 6, 12, and 24 h post-infection (hpi), and at 24 hpi, the number of DEGs was the highest. Furthermore, changes in the expression of interferons, which are mainly related to apoptosis and activation of the host innate immune pathway, were found in the PEDV and PDCoV infection and coinfection groups. Additionally, 43 ISGs, including GBP2, IRF1, ISG20, and IFIT2, were upregulated during PEDV or PDCoV infection. Furthermore, we found that ISG20 significantly inhibited PEDV and PDCoV infection in LLC-PK cells. The transcriptomic profiles of cells coinfected with PEDV and PDCoV were reported, providing reference data for understanding the host response to PEDV and PDCoV coinfection.

Development of multiplex cross displacement amplification combined with lateral flow biosensor assay for detection of virulent shigella sonnei.

Shigella sonnei is the most common Shigella spp. in developed areas and the second most common in undeveloped regions. In this study, a multiple cross displacement amplification (MCDA) assay was used in combination with a lateral flow biosensor (LFB) assay to detect virulent S. sonnei strains containing the ipaH and wbgX genes. The multiplex MCDA-LFB assay detected wbgX at ≥1 pg/µL and ipaH at ≥10 fg/µL within 30 min in pure cultures maintained at 63°C. This assay was sensitive for ~37 CFU of virulent S. sonnei and ~3.7 CFU of Shigella spp. and enteroinvasive E. coli in stimulated fecal samples and had 100% specificity among 59 reference strains. The MCDA-LFB assay was also able to differentiate between virulent S. sonnei and other Shigella spp. and enteroinvasive E. coli among 99 clinical isolates. In summary, a multiplex MCDA-LFB assay was developed for rapid, convenient, point-of-care, and accurate identification of virulent S. sonnei within 30 min and at a constant temperature without the need for expensive lab equipment.

Recognition method for the health risks of potentially toxic elements in a headwater catchment.

The spatial association of potentially toxic elements (PTEs) in soil-crop-groundwater systems is poorly recognised. In this study, the contents of arsenic (As), cadmium (Cd), copper (Cu) and lead (Pb) in paddy soils, rice and groundwater in the Xiancha River catchment were determined. The intrinsic effects of PTEs in soils on their spatial distribution in groundwater and rice were explored. Also, the potential sources and health risks of PTEs in multi-media were investigated. Results showed that the mean contents of As and Cd in soils were 23.86 and 0.26 mg kg-1, respectively. In groundwater, the maximum (average) content of As reached 6.55 (1.84) µg L-1. Moreover, As contents in soils and groundwater showed a sound spatial correlation (q = 0.81), and this is supported by the result of the soil column experiment, indicating homology and the strong vertical migration capacity of As. The non-homologous patterns of Pb, Cu and Cd contaminations in soil-groundwater system suggested that geogenic processes influenced the distribution of these PTEs. Cd presented a poor spatial correlation in soil-rice system, as multiple factors controlled its transfer process. Multivariate statistical analysis results demonstrated that As, Cu and Pb in soils mainly came from agricultural sources, whereas high Cd levels were from mining activities. Additionally, direct consumption of As-contaminated groundwater and Cd-contaminated rice posed significant health risks to local residents. This study, which proposes a risk recognition method used to investigate target PTEs in multi-media, may serve as a valuable reference for further research involving catchments.

[Construction of recombinant adenovirus expressing capsid protein of serotype O foot-and-mouth disease virus and analysis of its immunogenicity].

In order to construct a recombinant replication deficient human type 5 adenovirus (Ad5) expressing a foot-and-mouth disease virus (FMDV) capsid protein, specific primers for P12A and 3B3C genes of FMDV-OZK93 were synthesized. The P12A and 3B3C genes were then amplified and connected by fusion PCR, and a recombinant shuttle plasmid pDC316-mCMV-EGFP-P12A3B3C expressing the FMDV-OZK93 capsid protein precursor P12A and 3B3C protease were obtained by inserting the P12A3B3C gene into the pDC316-mCMV-EGFP plasmid. The recombinant adenovirus rAdv-P12A3B3C-OZK93 was subsequently packaged, characterized and amplified using AdMaxTM adenovirus packaging system, and the expression was verified by infecting human embryonic kidney cell HEK-293. The humoral and cellular immunity levels of well-expressed and purified recombinant adenovirus immunized mice were evaluated. The results showed that rAdv-P12A3B3C-OZK93 could be stably passaged and the maximum virus titer reached 1×109.1 TCID50/mL. Western blotting and indirect immunofluorescence showed that rAdv-P12A3B3C-OZK93 expressed the FMDV-specific proteins P12A and VP1 in HEK-293 cells. In addition, the PK cell infection experiment confirmed that rAdv-P12A3B3C-OZK93 could infect porcine cells, which is essential for vaccination in pigs. Comparing with the inactivated vaccine group, the recombinant adenovirus could induce higher FMDV-specific IgG antibodies, γ-IFN and IL-10. This indicates that the recombinant adenovirus has good immunity for animal, which is very important for the subsequent development of foot-and-mouth disease vaccine.

Comparative Genomic Analysis Reveals Potential Pathogenicity and Slow-Growth Characteristics of Genus Brevundimonas and Description of Brevundimonas pishanensis sp. nov.

The genus Brevundimonas consists of Gram-negative bacteria widely distributed in environment and can cause human infections. However, the genomic characteristics and pathogenicity of Brevundimonas remain poorly studied. Here, the whole-genome features of 24 Brevundimonas type strains were described. Brevundimonas spp. had relatively small genomes (3.13 ± 0.29 Mb) within the family Caulobacteraceae but high G+C contents (67.01 ± 2.19 mol%). Two-dimensional hierarchical clustering divided those genomes into 5 major clades, in which clades II and V contained nine and five species, respectively. Interestingly, phylogenetic analysis showed a one-to-one match between core and accessory genomes, which suggested coevolution of species within the genus Brevundimonas. The unique genes were annotated to biological functions like catalytic activity, signaling and cellular processes, multisubstance metabolism, etc. The majority of Brevundimonas spp. harbored virulence-associated genes icl, tufA, kdsA, htpB, and acpXL, which encoded isocitrate lyase, elongation factor, 2-dehydro-3-deoxyphosphooctonate aldolase, heat shock protein, and acyl carrier protein, respectively. In addition, genomic islands (GIs) and phages/prophages were identified within the Brevundimonas genus. Importantly, a novel Brevundimonas species was identified from the feces of a patient (suffering from diarrhea) by the analyses of biochemical characteristics, phylogenetic tree of 16S rRNA gene, multilocus sequence analysis (MLSA) sequences, and genomic data. The name Brevundimonas pishanensis sp. nov. was proposed, with type strain CHPC 1.3453 (= GDMCC 1.2503T = KCTC 82824T). Brevundimonas spp. also showed obvious slow growth compared with that of Escherichia coli. Our study reveals insights into genomic characteristics and potential virulence-associated genes of Brevundimonas spp., and provides a basis for further intensive study of the pathogenicity of Brevundimonas. IMPORTANCEBrevundimonas spp., a group of bacteria from the family Caulobacteraceae, is associated with nosocomial infections, deserve widespread attention. Our study elucidated genes potentially associated with the pathogenicity of the Brevundimonas genus. We also described some new characteristics of Brevundimonas spp., such as small chromosome size, high G+C content, and slow-growth phenotypes, which made the Brevundimonas genus a good model organism for in-depth studies of growth rate traits. Apart from the comparative analysis of the genomic features of the Brevundimonas genus, we also reported a novel Brevundimonas species, Brevundimonas pishanensis, from the feces of a patient with diarrhea. Our study promotes the understanding of the pathogenicity characteristics of Brevundimonas species bacteria.