Sequential Immunizations with Influenza Neuraminidase Protein Followed by Peptide Nanoclusters Induce Heterologous Protection.

Enhancing cross-protections against diverse influenza viruses is desired for influenza vaccinations. Neuraminidase (NA)-specific antibody responses have been found to independently correlate with a broader influenza protection spectrum. Here, we report a sequential immunization regimen that includes priming with NA protein followed by boosting with peptide nanoclusters, with which targeted enhancement of antibody responses in BALB/c mice to certain cross-protective B-cell epitopes of NA was achieved. The nanoclusters were fabricated via desolvation with absolute ethanol and were only composed of composite peptides. Unlike KLH conjugates, peptide nanoclusters would not induce influenza-unrelated immunity. We found that the incorporation of a hemagglutinin peptide of H2-d class II restriction into the composite peptides could be beneficial in enhancing the NA peptide-specific antibody response. Of note, boosters with N2 peptide nanoclusters induced stronger serum cross-reactivities to heterologous N2 and even heterosubtypic N7 and N9 than triple immunizations with the prototype recombinant tetrameric (rt) N2. The mouse challenge experiments with HK68 H3N2 also demonstrated the strong effectiveness of the peptide nanocluster boosters in conferring heterologous protection.

Antiviral effects of duck type I and type III interferons against Duck Tembusu virus in vitro and in vivo.

Duck Tembusu Virus (DTMUV) is a newly emerging avian flavivirus that causes substantial economic losses to the duck industry in Asia by causing severe egg drop syndrome and fatal encephalitis in domestic ducks. During viral replication, host cells recognize the RNA structures produced by DTMUV, which triggers the production of interferons (IFNs) to inhibit viral replication. However, the function of duck type I and type III IFNs in inhibiting DTMUV infection remains largely unknown. In this study, we expressed and purified recombinant duck IFN-ß (duIFN-ß) and IFN-λ (duIFN-λ) in Escherichia coli and evaluated their antiviral activity against vesicular stomatitis virus (VSV). Furthermore, we found that both duIFN-ß and duIFN-λ activated the ISRE promoter and induced the expression of ZAP, OAS, and RNaseL in duck embryo fibroblasts (DEFs). Notably, duIFN-ß showed faster and more potent induction of ISGs in vitro and in vivo compared to duIFN-λ. Moreover, both duIFN-ß and duIFN-λ showed high potential to inhibit DTMUV infection in DEFs, with duIFN-ß demonstrating better antiviral efficacy than duIFN-λ against DTMUV in ducks. In conclusion, our results revealed that both duIFN-ß and duIFN-λ can induce ISGs production and exhibit significant antiviral activity against DTMUV in vitro and in vivo, providing new insights for the development of antiviral therapeutic strategies in ducks.

Boost immunizations with NA-derived peptide conjugates achieve induction of NA inhibition antibodies and heterologous influenza protections.

Neuraminidase is suggested as an important component for developing a universal influenza vaccine. Targeted induction of neuraminidase-specific broadly protective antibodies by vaccinations is challenging. To overcome this, we rationally select the highly conserved peptides from the consensus amino acid sequence of the globular head domains of neuraminidase. Inspired by the B cell receptor evolution process, a reliable sequential immunization regimen is designed to result in immuno-focusing by steering bulk immune responses to a selected region where broadly protective B lymphocyte epitopes reside. After priming neuraminidase protein-specific antibody responses in C57BL/6 or BALB/c inbred mice strains by immunization or pre-infection, boost immunizations with certain neuraminidase-derived peptide-keyhole limpet hemocyanin conjugates significantly strengthened serum neuraminidase inhibition activities and cross-protections. Overall, this study provides proof of concept for a peptide-based sequential immunization strategy for achieving targeted induction of cross-protective antibody response, which provides references for designing universal vaccines against other highly variable pathogens.

A Library of Promoter-gfp Fusion Reporters for Studying Systematic Expression Pattern of Cyclic-di-GMP Metabolism-Related Genes in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa is an environmental microorganism and is a model organism for biofilm research. Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that plays critical roles in biofilm formation. P. aeruginosa contains approximately 40 genes that encode enzymes that participate in the metabolism of c-di-GMP (biosynthesis or degradation), yet it lacks tools that aid investigation of the systematic expression pattern of those genes. In this study, we constructed a promoter-gfp fusion reporter library that consists of 41 reporter plasmids. Each plasmid contains a promoter of corresponding c-di-GMP metabolism-related (CMR) genes from P. aeruginosa reference strain PAO1; thus, each promoter-gfp fusion reporter can be used to detect the promoter activity as well as the transcription of corresponding gene. The promoter activity was tested in P. aeruginosa and Escherichia coli. Among the 41 genes, the promoters of 26 genes showed activity in both P. aeruginosa and E. coli. The library was applied to determine the influence of different temperatures, growth media, and subinhibitory concentrations of antibiotics on the transcriptional profile of the 41 CMR genes in P. aeruginosa. The results showed that different growth conditions did affect the transcription of different genes, while the promoter activity of a few genes was kept at the same level under several different growth conditions. In summary, we provide a promoter-gfp fusion reporter library for systematic monitoring or study of the regulation of CMR genes in P. aeruginosa. In addition, the functional promoters can also be used as a biobrick for synthetic biology studies. IMPORTANCE The opportunistic pathogen P. aeruginosa can cause acute and chronic infections in humans, and it is one of the main pathogens in nosocomial infections. Biofilm formation is one of the most important causes for P. aeruginosa persistence in hosts and evasion of immune and antibiotic attacks. c-di-GMP is a critical second messenger to control biofilm formation. In P. aeruginosa reference strain PAO1, 41 genes are predicted to participate in the making and breaking of this dinucleotide. A major missing piece of information in this field is the systematic expression profile of those genes in response to changing environment. Toward this goal, we constructed a promoter-gfp transcriptional fusion reporter library that consists of 41 reporter plasmids, each of which contains a promoter of corresponding c-di-GMP metabolism-related genes in P. aeruginosa. This library provides a helpful tool to understand the complex regulation network related to c-di-GMP and to discover potential therapeutic targets.

Dual functions: A coumarin-chalcone conjugate inhibits cyclic-di-GMP and quorum-sensing signaling to reduce biofilm formation and virulence of pathogens.

Antibiotic resistance or tolerance of pathogens is one of the most serious global public health threats. Bacteria in biofilms show extreme tolerance to almost all antibiotic classes. Thus, use of antibiofilm drugs without bacterial-killing effects is one of the strategies to combat antibiotic tolerance. In this study, we discovered a coumarin-chalcone conjugate C9, which can inhibit the biofilm formation of three common pathogens that cause nosocomial infections, namely, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli, with the best antibiofilm activity against P. aeruginosa. Further investigations indicate that C9 decreases the synthesis of the key biofilm matrix exopolysaccharide Psl and bacterial second messenger cyclic-di-GMP. Meanwhile, C9 can interfere with the regulation of the quorum sensing (QS) system to reduce the virulence of P. aeruginosa. C9 treatment enhances the sensitivity of biofilm to several antibiotics and reduces the survival rate of P. aeruginosa under starvation or oxidative stress conditions, indicating its excellent potential for use as an antibiofilm-forming and anti-QS drug.

Chimeric Virus-like Particles Co-Displaying Hemagglutinin Stem and the C-Terminal Fragment of DnaK Confer Heterologous Influenza Protection in Mice.

Influenza virus hemagglutinin (HA) stem is currently regarded as an extremely promising immunogen for designing universal influenza vaccines. The appropriate antigen-presenting vaccine vector would be conducive to increasing the immunogenicity of the HA stem antigen. In this study, we generated chimeric virus-like particles (cVLPs) co-displaying the truncated C-terminal of DnaK from Escherichia coli and H1 stem or full-length H1 antigen using the baculovirus expression system. Transmission electronic micrography revealed the expression and presentation of H1 stem antigens on the surface of VLPs. Vaccinations of mice with the H1 stem cVLPs induced H1-specific immune responses and provided heterologous immune protection in vivo, which was more effective than vaccinations with VLPs displaying H1 stem alone in protecting mice against weight loss as well as increasing survival rates after lethal influenza viral challenge. The results indicate that the incorporation of the truncated C-terminal of DnaK as an adjuvant protein into the cVLPs significantly enhances the H1-specific immunity and immune protection. We have explicitly identified the VLP platform as an effective way of expressing HA stem antigen and revealed that chimeric VLP is an vaccine vector for developing HA stem-based universal influenza vaccines.

The Chinese knotting technique assist anatomical anterior cruciate ligament reconstruction for aggressive rehabilitation.

Aggressive rehabilitation after anterior cruciate ligament (ACL) reconstruction may result in better clinical outcomes and fewer complications such as knee stiffness and weakness. We explored the effect of the Chinese knotting technique (CKT) for aggressive rehabilitation after ACL reconstruction. Ninety-one anatomical ACL reconstruction cases from 2016 to 2020 were retrospectively reviewed. All patients were operated by the same senior physician and his team. According to the reconstruction with or without CKT, the patients were divided into 2 groups. Both groups received aggressive rehabilitation. The follow-up time of 91 patients was more than 2 years. In total, 43 out of the 91 patients were in the CKT group, and 48 were in the routine group. The knee joint kinematics recorded by Opti_Knee revealed no significant difference among the CKT group, the routine group, and healthy adults at 3, 6, 12, and 24 months after the operation, respectively. The internal and external rotation angle and the anteroposterior displacement at 3 and 6 months after the operation in the CKT group were smaller than in the routine group and were similar to that of the healthy adults. There was no significant difference in flexion and extension angle, varus or valgus angle, proximal-distal displacement, or the internal or external displacement between the 2 groups. In addition, there was no significant difference in 6 degrees of freedom of the knee between the 2 groups at 12 and 24 months after the operation, respectively, which was similar to healthy adults. Compared to the routine group, the International Knee Documentation Committee scores were significantly higher in the CKT group at the 3, 6, and 12 months, respectively, but no difference was observed at 24 months (P = .749). The Lysholm score was significantly higher in the CKT group at the 3 and 6 months postoperatively, while there was no difference at 12 and 24 months, respectively. In short-term observation, the ACL reconstruction with CKT, which can sustain aggressive rehabilitation and prevent the loosening of ACL graft, can lead to better clinical outcomes and kinematics recovery of the knee compared to routine technique.

[Research progress of internal tension relieving technique in assisting anterior cruciate ligament reconstruction].

OBJECTIVE: To review the research progress of internal tension relieving technique in assisting anterior cruciate ligament (ACL) reconstruction with tendon grafts. METHODS: The in vivo and in vitro biomechanical tests, animal experiments, and clinical studies on the use of internal tensioning relieving technique assisted ACL reconstruction in recent years were extensively reviewed, the impact of this technology on the biomechanics, histological changes of grafts, and the clinical effectiveness were analyzed and summarized. RESULTS: The internal tensioning relieving technique based on non-absorbable high-strength sutures can reduce the risk of relaxation and rupture by enhancing the biomechanical strength of tendon grafts in vitro and in vivo, it shows good biocompatibility and support for the ligamentation of the tendon grafts and the establishment of the direct tendon-bone interface in terms of histology. This technique improves postoperative initial joint stability, range of motion, and functional scores in clinical practic, when combining with the enhanced recovery after surgery can effectively promote patients to return to pre-injury exercise level without serious complications. CONCLUSION: The preliminary research results have confirmed the efficacy and safety of the internal tension relieving technique on assisting ACL reconstruction, then showes some degree of significance and prospect, but more research is needed to further optimize tension-relieving devices and related surgical techniques, and clarify the specific effects of this technique on graft's structure remodeling, biomechanical function, and long-term clinical results.

virusMED: an atlas of hotspots of viral proteins.

Metal binding sites, antigen epitopes and drug binding sites are the hotspots in viral proteins that control how viruses interact with their hosts. virusMED (virus Metal binding sites, Epitopes and Drug binding sites) is a rich internet application based on a database of atomic interactions around hotspots in 7041 experimentally determined viral protein structures. 25306 hotspots from 805 virus strains from 75 virus families were characterized, including influenza, HIV-1 and SARS-CoV-2 viruses. Just as Google Maps organizes and annotates points of interest, virusMED presents the positions of individual hotspots on each viral protein and creates an atlas upon which newly characterized functional sites can be placed as they are being discovered. virusMED contains an extensive set of annotation tags about the virus species and strains, viral hosts, viral proteins, metal ions, specific antibodies and FDA-approved drugs, which permits rapid screening of hotspots on viral proteins tailored to a particular research problem. The virusMED portal (https://virusmed.biocloud.top) can serve as a window to a valuable resource for many areas of virus research and play a critical role in the rational design of new preventative and therapeutic agents targeting viral infections.

Glycosylation on envelope glycoprotein of duck Tembusu virus affects virus replication in vitro and contributes to the neurovirulence and pathogenicity in vivo.

Duck Tembusu virus (DTMUV), an emergent flavivirus, causes domestic waterfowls to suffer from severe egg-drop syndrome and fatal encephalitis, greatly threatens duck production globally. Like other mosquito-borne flaviviruses, the envelope (E) protein of all DTMUV strains was N-glycosylated at the amino acid position 154. Thus far, the biological roles of DTMUV E glycosylation have remained largely unexplored. Herein, we demonstrated the key roles of E glycosylation in the replication and pathogenicity of DTMUV in ducks by characterizing the reverse-genetics-derived DTMUV wild-type MC strain and MC bearing mutations (N154Q and N154I) that abolish the E glycosylation. Our data showed that the disruption of E glycosylation could substantially impair virus attachment, entry, and infectivity in DEFs and C6/36 cells. Notably, ducks inoculated intracerebrally with the wild-type virus exhibited severe disease onset. In contrast, those inoculated with mutant viruses were mildly affected as manifested by minimal weight loss, no mortality, lower viral loads in the various tissues, and reduced brain lesions. Attenuated phenotypes of the mutant viruses might be partly associated with lower inflammatory cytokines expression in the brains of infected ducks. Our study offers the first evidence that E glycosylation is vital for DTMUV replication, pathogenicity, and neurovirulence in vivo.

A novel method to establish the rabbit model of knee osteoarthritis: intra-articular injection of SDF-1 induces OA.

BACKGROUND: Animal model of Knee Osteoarthritis (OA) is the primary testing methodology for studies on pathogenic mechanisms and therapies of human OA disease. Recent major modeling methods are divided into artificially induced and spontaneous. However, these methods have some disadvantages of slow progression, high cost and no correlation with the pathogenesis of OA. METHODS: Our studies attempted to find a rapid, easy, and consistent with the natural pathological process of OA modeling method by intra-articular injection of stromal cell-derived factor 1 (SDF-1) in the rabbit knee. After induction we collected cartilage specimens from the medial femoral condyle to undergo macroscopic, histological, immunohistochemical, and biochemical evaluations. Meanwhile, compared with Hulth surgical method to evaluate its efficacy. RESULTS: Macroscopic observation and modified Mankin score of histological staining exhibited typical features of middle stage OA cartilage in SDF-1 injected groups. Immunohistochemically, the positive expression of interleukin-1 (IL-1) and tumor necrosis factor α(TNF-α) was earlier and higher in high dose SDF-1 group than the surgical group. The matrix metalloproteinases (MMPs) in synovial fluid and chondrocytes significantly increased, but type II collagen (COLII) and aggrecan (ACAN) protein expressions decreased in SDF-1 injected group following the extension of time and increase of SDF-1 concentration. CONCLUSIONS: Our data indicated intra-articular injection of SDF-1 (40µg/kg, three times for 12 weeks) can induce rabbit knee OA model successfully more rapidly and easily than traditional surgical modeling. The study provided a further option for the establishment of knee OA animal model.

[Research progress in femoral tunnel positioning points of medial patellofemoral ligament reconstruction].

OBJECTIVE: To review the research progress of location methods and the best femoral insertion position of medial patellofemoral ligament (MPFL) reconstruction of femoral tunnel, and provide reference for surgical treatment. METHODS: The literature about femoral insertion position of the MPFL reconstruction in recent years was extensively reviewed, and the anatomical and biomechanical characteristics of MPFL, as well as the advantages and disadvantages of femoral tunnel positioning methods were summarized. RESULTS: The accurate establishment of the femoral anatomical tunnel is crucial to the success of MPFL reconstruction. At present, there are mainly two kinds of methods for femoral insertion: radiographic landmark positioning method and anatomical landmark positioning method. Radiographic landmark positioning method has such advantages as small incision and simple operation, but it can not be accurately positioned for patients with severe femoral trochlear dysplasia. It is suggested to combine with the anatomical landmark positioning method. These methods have their own advantages and disadvantages, and there is no unified positioning standard. In recent years, the use of three-dimensional design software can accurately assist in the MPFL reconstruction, which has become a new trend. CONCLUSION: Femoral tunnel positioning of the MPFL reconstruction is very important. The current positioning methods have their own advantages and disadvantages. Personalized positioning is a new trend and has not been widely used in clinic, its effectiveness needs further research and clinical practice and verification.

Construction of tissue engineering bone with the co­culture system of ADSCs and VECs on partially deproteinized biologic bone in vitro: A preliminary study.

Scaffold­based bone tissue engineering has therapeutic potential in the regeneration of osseous defects. The present study aimed to explore the adhesion and cell viability of a co­culture system composed of vascular endothelial cells PI­/Annexin V+ represents early apoptotic cells, and PI+/Annexin V+ represents late apoptotic cells (VECs) and adipose­derived stem cells (ADSCs) on partially deproteinized biologic bone (PDPBB) in vitro, and determine the optimum time period for maximum cell viability that could possibly be used for standardizing the scaffold transplant into the in vivo system. VECs and ADSCs were isolated from pregnant Sprague­Dawley rats and confirmed by immunostaining with von Willebrand factor and CD90, respectively. PDPBB was prepared using standardized protocols involving coating partially deproteinized bone with fibronectin. PDPBB was incubated in a mono­culture with VECs or ADSCs, or in a co­culture with both of these cells at a ratio of 1:1. An MTT assay was used to assess the adhesion and cell viability of VECs and ADSCs on PDPBB in the three different cultures. Scanning electron microscopy was used to observe the adhesion, cell viability and morphology of the different types of cells on PDPBB. It was observed that the absorbance of each group increased gradually and peaked on the 10th day; the highest absorbance was found for the co­cultured cells group. The difference of cell viability between each cell group was statistically significant. On the 10th day, in the co­cultured cells group, several cells adhered on the PDPBB material and a nest­like distribution morphology was observed. Therefore, the adhesion and cell viability of the co­cultured cells was higher compared with the mono­cultures of VECs or ADSCs. As cell viability was highest on the 10th day, this could be the optimal length of time for incubation and therefore could be used for in vivo experiments.

The use of a 3D-printed individualized navigation template to assist in the anatomical reconstruction surgery of the anterior cruciate ligament.

BACKGROUND: To explore the location accuracy and early clinical outcomes of using a 3D-printed individualized navigation template to assist in the reconstruction of the anterior cruciate ligament (ACL). METHODS: A single center randomized control study was conducted. Patients with ACL injury were treated with a conventional operation or an operation assisted by a 3D-printed individualized navigation template (the 3D group). The primary endpoint was the accuracy of the actual reconstruction compared with the planned position. RESULTS: There were 20 and 23 participants in the conventional group and the 3D group, respectively. There were no differences in the bone tunnel position between the actual postoperative position and the preoperative design in the 3D group (P>0.05). Compared with the 3D group, the positioning of the femoral tunnel was more inferior and shallower in the conventional group (P<0.05). The position of the tibia tunnel was closer to the anterior and medial edge of the tibial platform in the conventional group compared to the 3D group (P<0.05). The intraoperative positioning time was shorter in the 3D group than in the conventional group (3.3±1.0 vs. 5.9±1.8 minutes, P<0.001). The Lysholm and International Knee Documentation Committee scores did not differ between the two groups (P>0.05 for both), and all patients improved after surgery (P<0.001). CONCLUSIONS: The 3D-printed individualized navigation template showed good location accuracy and resulted in reduced intraoperative positioning time compared to the traditional method for ACL reconstruction.

Molecular cloning and functional characterization of duck TYK2.

Tyrosine kinase 2 (TYK2), a member of Janus kinase family, has been identified as a crucial protein in signal transduction initiated by interferons or interleukins in mammals. However, the function of avian TYK2 in innate immune response remains largely unknown. In this study, the full-length duck TYK2 (duTYK2) cDNA was cloned for the first time, which encoded a putative protein of 1187 amino acid residues and showed the high sequence similarity with bald eagle, crested ibis, and white-tailed tropicbird TYK2s. The duTYK2 was widely expressed in all examined tissues of healthy ducks and showed diffuse cytoplasmic localization in duck embryo fibroblasts (DEFs). Overexpression of duTYK2 significantly enhanced ISRE promoter activity and induced the expression of viperin, PKR, 2',5'-OAS, Mx and ZAP in DEFs. The C-terminal kinase domain of duTYK2 is essential for duTYK2-mediated ISRE promoter activation. Furthermore, knockdown of duTYK2 dramatically decreased duck Tembusu virus (DTMUV)-, duck enteritis virus (DEV)-, poly(I:C)- or poly(dA:dT)-induced ISRE promoter activation. Additionally, duTYK2 expression exhibited antiviral activity against DTMUV. These results indicated that duTYK2 played a critical role in duck antiviral innate immunity.

Molecular cloning and functional characterization of duck Janus kinase 1.

Janus kinase 1 (JAK1) is a member of JAK family of non-receptor protein tyrosine kinases that plays critical roles in transducing cytokine signals via JAK-signal transducer and activator of transcription (STAT) signaling pathway. The importance of JAK1 in innate immunity has been well-studied in mammals and fish, yet in avian remains largely unknown. Here, we cloned the full-length of the duck JAK1 (duJAK1) gene for the first time. DuJAK1 encoded a protein of 1152 amino acids and possessed high amino acid identity with goose and budgerigar JAK1s. The duJAK1 was expressed in all detected tissues, especially high in the thymus and bursa of Fabricius. Overexpression of duJAK1 significantly activated ISRE promoter activity and induced duck viperin, 2', 5'-OAS, MX, PKR and ZAP expression. Knockdown of duJAK1 by small interfering RNA significantly inhibited duck Tembusu virus (DTMUV)-, duck Enteritis virus (DEV)-, poly (I:C)-, poly (dA:dT)- or Sendai virus (SeV)-induced ISRE promoter activation. Furthermore, duJAK1 exhibited antiviral activity against DTMUV infection. These results will help us understand the function of JAK family proteins in duck antiviral immunity.

Genetic manipulation of porcine deltacoronavirus reveals insights into NS6 and NS7 functions: a novel strategy for vaccine design.

Porcine deltacoronavirus (PDCoV) is an emerging swine coronavirus that causes severe diarrhea, resulting in high mortality in neonatal piglets. Despite widespread outbreaks in many countries, no effective PDCoV vaccines are currently available. Here, we generated, for the first time, a full-length infectious cDNA clone of PDCoV. We further manipulated the infectious clone by replacing the NS6 gene with a green fluorescent protein (GFP) to generate rPDCoV-ΔNS6-GFP; likewise, rPDCoV-ΔNS7 was constructed by removing the ATG start codons of the NS7 gene. Growth kinetics studies suggest that rPDCoV-ΔNS7 could replicate similarly to that of the wild-type PDCoV, whereas rPDCoV-ΔNS6-GFP exhibited a substantial reduction of viral titer in vitro and in vivo. Piglets inoculated with rPDCoV-ΔNS7 or wild-type PDCoV showed similar diarrheic scores and pathological injury. In contrast, rPDCoV-ΔNS6-GFP-infected piglets did not show any clinical signs, indicating that the NS6 protein is an important virulence factor of PDCoV and that the NS6-deficient mutant virus might be a promising live-attenuated vaccine candidate. Taken together, the reverse genetics platform described here not only provides more insights into the role of PDCoV accessory proteins in viral replication and pathogenesis, but also allows the development of novel vaccines against PDCoV infection.

Controlled Growth of Ultra-Thick Polymer Brushes via Surface-Initiated Atom Transfer Radical Polymerization with Active Polymers as Initiators.

Polymer brushes exhibit functionalities useful for a large number of applications. Often these functionalities only emerge when the polymer brushes have a desired thickness. Here, a significant breakthrough is achieved in the synthesis of ultra-thick polymer brushes using polymer initiators in the approach of surface-initiated atom transfer radical polymerization, yielding polymer brushes with a controllable thickness up to 15.1 µm. This is reportedly the thickest polymer brush ever synthesized. This approach is applicable for several monomers such as acrylonitrile, methyl acrylate, and styrene, and for other types of polymer substrates such as fibers.

Cloning, expression, and characterization of a novel nitrilase, PaCNit, from Pannonibacter carbonis Q4.6.

OBJECTIVE: Identification of a heavy metal ion-stimulated nitrilase with broad-spectrum substrate specificity. RESULTS: A novel nitrilase, PaCNit, was identified from Pannonibacter carbonis Q4.6 and its enzymatic properties were investigated. The maximum activity of PaCNit was observed at 65 °C and pH 7.0. PaCNit showed broad substrate specificity towards aliphatic, aromatic, and heterocyclic nitriles, and was tolerant to different organic solvents. Remarkably, PaCNit activity was highly stimulated by metal ions, particularly by Ag+ and Hg2+. CONCLUSION: PaCNit nitrilase has a broad range of substrate specificity and can be activated by heavy metal ions. This specific characteristic makes it have a great potential for industrial application.

Genomic characterization and pathogenicity of porcine deltacoronavirus strain CHN-HG-2017 from China.

Porcine deltacoronavirus (PDCoV) was first detected in Hong Kong and has recently spread to many countries around the world. PDCoV causes acute diarrhea and vomiting in pigs, resulting in significant economic losses in the global pork industry. In this study, a Chinese PDCoV strain, designated CHN-HG-2017, was isolated from feces of a suckling piglet with severe watery diarrhea on a farm located in central China. Subsequently, the virus was identified by an indirect immunofluorescence assay and electron microscopy. A nucleotide sequence alignment showed that the whole genome of CHN-HG-2017 is 97.6%-99.1% identical to other PDCoV strains. Analysis of potential recombination sites showed that CHN-HG-2017 is a possible recombinant originating from the strains CH/SXD1/2015 and Vietnam/HaNoi6/2015. Furthermore, the pathogenicity of this recombinant PDCoV strain was investigated in 5-day-old piglets by oral inoculation. The challenged piglets developed typical symptoms, such as vomiting, anorexia, diarrhea and lethargy, from 1 to 7 days post-inoculation (DPI). Viral shedding was detected in rectal swabs until 14 DPI in the challenged piglets. Interestingly, high titers of virus-neutralizing antibodies in sera were detected at 21 DPI. Tissues of small intestines from CHN-HG-2017-infected piglets at 4 DPI displayed significant macroscopic and microscopic lesions with clear viral antigen expression. Our analysis of the full genome sequence of a recombinant PDCoV and its virulence in suckling piglets might provide new insights into the pathogenesis of PDCoV and facilitate further investigation of this newly emerged pathogen.