Development of a droplet digital PCR method for detection of porcine circovirus 4.

BACKGROUND: Porcine circovirus 4 (PCV4), a newly emerging virus that was first discovered in 2019, may pose a potential threat to the pig industry. Droplet digital PCR (ddPCR) is an absolute quantitative method that has high sensitivity and accuracy. In this study, we developed a novel ddPCR assay to detect PCV4. Furthermore, we evaluated the detection limit, sensitivity, specificity and reproducibility of the ddPCR and TaqMan real-time quantitative PCR (qPCR) and tested 160 clinical samples to compare the detection rate of the two methods. RESULTS: The detection limit for ddPCR was 0.54 copies/µL, 10.6 times greater sensitivity than qPCR. Both ddPCR and qPCR assays exhibited good linearity and repeatability, and the established ddPCR method was highly specific for PCV4. The results of clinical sample testing showed that the positivity rate of ddPCR (5.6%) was higher than that of qPCR (4.4%). CONCLUSIONS: This study successfully developed a sensitive, specific and repeatable ddPCR assay for PCV4 detection, which can be widely used in clinical diagnosis of PCV4 infections.

Nanomaterials-Functionalized Hydrogels for the Treatment of Cutaneous Wounds.

Hydrogels have been utilized extensively in the field of cutaneous wound treatment. The introduction of nanomaterials (NMs), which are a big category of materials with diverse functionalities, can endow the hydrogels with additional and multiple functions to meet the demand for a comprehensive performance in wound dressings. Therefore, NMs-functionalized hydrogels (NMFHs) as wound dressings have drawn intensive attention recently. Herein, an overview of reports about NMFHs for the treatment of cutaneous wounds in the past five years is provided. Firstly, fabrication strategies, which are mainly divided into physical embedding and chemical synthesis of the NMFHs, are summarized and illustrated. Then, functions of the NMFHs brought by the NMs are reviewed, including hemostasis, antimicrobial activity, conductivity, regulation of reactive oxygen species (ROS) level, and stimulus responsiveness (pH responsiveness, photo-responsiveness, and magnetic responsiveness). Finally, current challenges and future perspectives in this field are discussed with the hope of inspiring additional ideas.

Incorporation of a truncated form of flagellin (TFlg) into porcine circovirus type 2 virus-like particles enhances immune responses in mice.

BACKGROUND: Porcine circovirus type 2 (PCV2) is an economically important pathogen in the swine industry worldwide. Vaccination remains the principal tool to control PCV2-associated diseases (PCVADs). Current vaccines do not eliminate viral shedding in the environment. To enhance the efficacy of PCV2 vaccines, recombinant virus-like particles (VLPs) of PCV2 were generated by fusing a truncated form of flagellin FliC (TFlg: 85-111aa) with the PCV2 capsid protein (Cap). RESULTS: The recombinant proteins were expressed in Escherichia coli and detected using Western blotting. The abilities of the recombinant proteins to assemble into VLPs were observed under transmission electron microscopy (TEM). The protective immune responses of recombinant VLPs were further evaluated by immunization of mice. The results showed that insertion of TFlg into C terminal of the Cap protein did not affect the formation of VLPs and boosted both humoral and cellular immune responses in mice. After a challenge with PCV2, in the Cap-TFlg vaccinated group, viremia was milder and viral loads were lower as compared with those in the Cap vaccinated group. CONCLUSION: These results suggest that recombinant VLPs of PCV2 containing a TFlg adjuvant can be used as a promising PCV2 vaccine candidate.

Study of the immunogenicity of the VP2 protein of canine parvovirus produced using an improved Baculovirus expression system.

BACKGROUND: Canine parvovirus (CPV) is now recognized as a serious threat to the dog breeding industry worldwide. Currently used CPV vaccines all have their specific drawbacks, prompting a search for alternative safe and effective vaccination strategies such as subunit vaccine. VP2 protein is the major antigen targeted for developing CPV subunit vaccine, however, its production in baculovirus expression system remains challenging due to the insufficient yield. Therefore, our study aims to increase the VP2 protein production by using an improved baculovirus expression system and to evaluate the immunogenicity of the purified VP2 protein in mice. RESULTS: The results showed that high-level expression of the full length VP2 protein was achieved using our modified baculovirus expression system. The recombinant virus carrying two copies of VP2 gene showed the highest expression level, with a productivity of 186 mg/L, which is about 1.4-1.6 fold that of the recombinant viruses carrying only one copy. The purified protein reacted with Mouse anti-His tag monoclonal antibody and Rabbit anti-VP2 polyclonal antibody. BALB/c mice were intramuscularly immunized with purified VP2 protein twice at 2 week intervals. After vaccination, VP2 protein could induce the mice produce high level of hemagglutination inhibition antibodies. CONCLUSIONS: Full length CPV VP2 protein was expressed at high level and purified efficiently. Moreover, it stimulated mice to produce high level of antibodies with hemmaglutination inhibition properties. The VP2 protein expressed in this study could be used as a putative economic and efficient subunit vaccine against CPV infection.

Surface displaying of swine IgG1 Fc enhances baculovirus-vectored vaccine efficacy by facilitating viral complement escape and mammalian cell transduction.

Baculovirus-mediated gene transfer has been developed as a vaccine design strategy against a number of diseases without apparent viral replication. However, it has been hampered by complement-dependent inactivation, thus hindering the in vivo application of baculovirus. A variety of approaches have been exploited to bypass the complement system in the serum. In this study, we constructed and screened a series of baculovirus vectors displaying complement interfering factors, of which a baculovirus vector displaying swine IgG1 Fc (pFc) showed the highest complement antagonism (75.6%). Flow cytometry analysis of transduced cells demonstrated that the baculovirus display of pFc had a significant increase in transduction efficiency and transgene expression of reporter genes. On this basis, a VSV-G-pseudotyped with swine IgG1 Fc surface displayed baculovirus vector was developed to express the classical swine fever virus (CSFV) E2 gene. The translational enhancers Syn21 and P10UTR were incorporated to improve the antigen expression. The E2 gene was efficiently expressed in both insect and mammalian cells. Pigs immunized with this recombinant baculovirus developed high levels of E2-specific antibody, CSFV-specific neutralizing antibody and IFN-γ-secreting cellular immune responses. These results demonstrate that the strategy of surface-displaying swine IgG1 Fc has a great potential to improve the efficiency of baculovirus-vectored vaccine for CSFV and other swine pathogens.

Functional analysis of RNAi suppressor P19 on improving baculovirus yield and transgene expression in Sf9 cells.

OBJECTIVE: To investigate whether RNA interference suppressor P19 derived from tombusvirus can enhance baculovirus yield and transgene expression. RESULTS: A number of recombinant baculoviruses with P19 under the control of white spot syndrome virus immediate-early promoter, baculovirus early-to-late promoter, or P10 late promoter were constructed The budded virus titer and the expression levels of eGFP and luciferase were determined. P19 was clearly functional in Sf9 cells and could enhance baculovirus yield, eGFP and luciferase expression levels up to 6.8-, 1.8-, and 2.1-fold respectively, at 72 h post infection. CONCLUSION: P19 enhanced baculovirus production and transgene expression, and thus has potential applications in baculovirus-based gene therapy and vaccine studies.

Enhanced production of porcine circovirus type 2 (PCV2) virus-like particles in Sf9 cells by translational enhancers.

OBJECTIVE: To investigate the effect of three translational enhancers for enhancing transgene expression in baculovirus expression vector system using GFP as a reporter gene and selected translational enhancers to increase porcine circovirus type 2 (PCV2) VLPs production. RESULTS: P10UTR (the 3'-untranslated region from the baculovirus p10 gene), Syn21 (a synthetic AT-rich 21-bp sequence) and P10UTR/Syn21 increased the GFP yield by 1.4-, 4- and 4.8-fold, respectively. While IVS (intron from Drosophila myosin heavy chain gene) decreased the GFP yield by 65%. Moreover, the synergy of P10UTR/Syn21 increased the yield of PCV2 VLPs by 4.1 fold (45 µg/10(6) cells) compared with standard baculovirus vector. CONCLUSION: The synergy of P10UTR/Syn21 is a potential strategy to improve the recombinant vaccine production besides PCV2 VLPs in BEVS.

Identification of two novel linear epitopes on the E165R protein of African swine fever virus recognized by monoclonal antibodies.

African swine fever (ASF) is a highly fatal infectious disease in pigs, caused by the African swine fever virus (ASFV). It is characterized by short disease duration and high morbidity and mortality. In August 2018, ASF was first reported in China and it subsequently spread rapidly throughout the country, causing serious economic losses for the Chinese pig industry. Early detection plays a critical role in preventing and controlling ASF because there is currently no effective vaccine or targeted therapeutic medication available. Additionally, identifying conserved protective antigenic epitopes of ASFV is essential for the development of diagnostic reagents. The E165R protein, which is highly expressed in the early stages of ASFV infection, can serve as an important indicator for early detection. In this study, we successfully obtained high purity soluble prokaryotic expression of the E165R protein. We then utilized the purified recombinant E165R protein for immunization in mice to prepare monoclonal antibodies (mAbs) using the hybridoma fusion technique. After three subclonal screens, we successfully obtained three mAbs against ASFV E165R protein in cells named 1B7, 1B8, and 10B8. Through immunofluorescence assay (IFA) and Western blot, we confirmed that the prepared mAbs specifically recognize the baculovirus-expressed E165R protein. By using overlapping truncated E165R protein and overlapping peptide scanning analysis, we tentatively identified two novel linear B cell epitopes (13EAEAYYPPSV22 and 55VACEHMGKKC64) that are highly conserved in genotype I and genotype II of ASFV. Thus, as a detection antibody, it has the capability to detect ASFV across a wide range of genotypes, providing valuable information for the development of related immunodiagnostic reagents.

From Fluorescence-Transfer-Lightening-Printing-Assisted Conductive Adhesive Nanocomposite Hydrogels toward Wearable Interactive Optical Information-Electronic Strain Sensors.

The interactive flexible device, which monitors the human motion in optical and electrical synergistic modes, has attracted growing attention recently. The incorporation of information attribute within the optical signal is deemed advantageous for improving the interactive efficiency. Therefore, the development of wearable optical information-electronic strain sensors holds substantial promise, but integrating and synergizing various functions and realizing strain-mediated information transformation keep challenging. Herein, an amylopectin (AP) modified nanoclay/polyacrylamide-based nanocomposite (NC) hydrogel and an aggregation-induced-emission-active ink are fabricated. Through the fluorescence-transfer printing of the ink onto the hydrogel film in different strains with nested multiple symbolic information, a wearable interactive fluorescent information-electronic strain sensor is developed. In the sensor, the nanoclay plays a synergistic "one-stone-three-birds" role, contributing to "lightening" fluorescence (≈80 times emission intensity enhancement), ionic conductivity, and excellent stretchability (>1000%). The sensor has high biocompatibility, resilience (elastic recovery ratio: 97.8%), and strain sensitivity (gauge factor (GF): 10.9). Additionally, the AP endows the sensor with skin adhesiveness. The sensor can achieve electrical monitoring of human joint movements while displaying interactive fluorescent information transformation. This research poses an efficient strategy to develop multifunctional materials and provides a general platform for achieving next-generation interactive devices with prospective applications in wearable devices, human-machine interfaces, and artificial intelligence.

Genomic characterisation of a lentogenic Newcastle disease virus strain HX01 isolated from sick pigs in China.

This paper describes the complete genome sequence of HX01, an isolate of the Newcastle disease virus (NDV) collected from a swine disease outbreak. The genome is 15,186 nt long and consists of six genes in the order of 3'-NP-P-M-F-HN-L-5'. This genome has the same length as the old NDV genotypes (I-IV), whereas the new NDV genotypes (V-IX) are 15,192 nt long. Compared with the genomic sequences of the reference NDV strains, the HX01 genome is highly similar to the genome of other NDV strains. However, some unique features of the HN gene were found in HX01. HX01 possesses the motif (112)G-R-Q-G-R-L(117) at the fusion protein cleavage site, which is typical of lentogenic strains. Pathogenicity tests based on the mean death time and the intracerebral pathogenicity index also revealed the isolate's lentogenic character. Phylogenetic analysis based on the variable region of the F gene (nt 47-420) revealed that HX01 was clustered to genotype II within class II NDV. Genetically, HX01 has a high similarity with the La Sota vaccine strain based on the single gene or complete genomic but is far different from the prevalent genotype VIId NDV which circulates in fowls and waterfowls in mainland China.

Porcine Deltacoronavirus-like Particles Produced by a Single Recombinant Baculovirus Elicit Virus-Specific Immune Responses in Mice.

Porcine deltacoronavirus (PDCoV) causes diarrhea and vomiting in neonatal piglets worldwide and has the potential for cross-species transmission. Therefore, virus-like particles (VLPs) are promising vaccine candidates because of their safety and strong immunogenicity. To the best of our knowledge, the present study reported for the first time the generation of PDCoV VLPs using a baculovirus expression vector system, and electron micrograph analyses revealed that PDCoV VLPs appeared as spherical particles with a diameter similar to that of the native virions. Furthermore, PDCoV VLPs effectively induced mice to produce PDCoV-specific IgG and neutralizing antibodies. In addition, VLPs could stimulate mouse splenocytes to produce high levels of cytokines IL-4 and IFN-γ. Moreover, the combination of PDCoV VLPs and Freund's adjuvant could improve the level of the immune response. Together, these data showed that PDCoV VLPs could effectively elicit humoral and cellular immunity in mice, laying a solid foundation for developing VLP-based vaccines to prevent PDCoV infections.

Development of a droplet digital PCR assay for detection of group A porcine rotavirus.

Group A porcine rotavirus (PoRVA) is an important pathogen of acute enteritis in piglets, which has caused severe economic losses in the pig industry worldwide. A convenient, sensitive and specific diagnosis method is an urgent requirement for the surveillance of the PoRVA circulating in clinical samples. In this study, a novel and convenient droplet digital PCR (ddPCR) for the detection of PoRVA was developed using the conserved region of the VP6 gene. The detection limit of ddPCR was 1.81 ± 0.14 copies/rection, ~10 times greater sensitivity than TaqMan real-time quantitative PCR (qPCR). Both ddPCR and qPCR assays exhibited good linearity and repeatability, and the established ddPCR method was highly specific for PoRVA. The results of clinical sample testing showed that the positivity rate of ddPCR (5.6%) was higher than that of qPCR (4.4%). Therefore, the newly developed ddPCR assay could be widely used in clinical diagnosis of PoRVA infections.

Isolation and characterization of porcine epidemic diarrhea virus with a novel continuous mutation in the S10 domain.

Porcine epidemic diarrhea virus (PEDV), which re-emerged in China in 2010, has caused severe economic losses to the global pig industry. In this study, a PEDV strain, designated PEDV WMB, was isolated from piglets with severe diarrhea on a pig farm in Henan Province of China. Whole-genome sequencing and analysis revealed that the PEDV WMB strain belongs to subtype G2c and has a unique continuous mutation in the S10 antigenic epitope of the S protein. Moreover, the virus-neutralization (VN) test indicated that polyclonal antibodies against the S10 protein of other G1 and G2 strains showed reduced VN reactivity to PEDV WMB. The pathogenicity of PEDV WMB was further investigated in 3 day-old piglets. PEDV infection-related clinical symptoms and morphological lesions were observed and confirmed by histopathological and immunohistochemical examination (IHC). These results illustrated that continuous mutation of the S10 epitope might affect the immunogenicity or pathogenicity of PEDV, providing evidence of the need to monitor the genetic diversity of the virus and develop effective measures to prevent and control PEDV.

Sprayed PAA-CaO2 nanoparticles combined with calcium ions and reactive oxygen species for antibacterial and wound healing.

The most common socioeconomic healthcare issues in clinical are burns, surgical incisions and other skin injuries. Skin lesion healing can be achieved with nanomedicines and other drug application techniques. This study developed a nano-spray based on cross-linked amorphous calcium peroxide (CaO2) nanoparticles of polyacrylic acid (PAA) for treating skin wounds (PAA-CaO2 nanoparticles). CaO2 serves as a 'drug' precursor, steadily and continuously releasing calcium ions (Ca2+) and hydrogen peroxide (H2O2) under mildly acidic conditions, while PAA-CaO2 nanoparticles exhibited good spray behavior in aqueous form. Tests demonstrated that PAA-CaO2 nanoparticles exhibited low cytotoxicity and allowed L929 cells proliferation and migration in vitro. The effectiveness of PAA-CaO2 nanoparticles in promoting wound healing and inhibiting bacterial growth in vivo was assessed in SD rats using full-thickness skin defect and Staphylococcus aureus (S.aureus)-infected wound models based thereon. The results revealed that PAA-CaO2 nanoparticles demonstrated significant advantages in both aspects. Notably, the infected rats' skin defects healed in 12 days. The benefits are linked to the functional role of Ca2+ coalesces with H2O2 as known antibacterial and healing-promoted agents. Therefore, we developed nanoscale PAA-CaO2 sprays to prevent bacterial development and heal skin lesions.

Glucose determination behaviour of gold microspheres-electrodeposited carbon cloth flexible electrodes in neutral media.

Despite numerous advances in the field of nonenzymatic glucose detection, monitoring glucose in physiological applications is still a challenge and is mostly limited to electrode surface modification. This study proposes a simple method for electrodepositing cotton-like gold microspheres (CGMs) on a carbon cloth (CC) flexible electrode, with the potential for the functional supporting substrate to monitor glucose in a neutral environment. It was demonstrated that the voltammetric response of glucose oxidation increased with increases in glucose concentration in the 3D functional flexible substrate; moreover, the amperometric response of glucose oxidation increased over time. The results indicate that the functional flexible electrode-made of gold microspheres-based carbon cloth with a predefined geometry and pore-architecture network to promote the medium-permeation and synergetic effects between CGMs and CC-can be a suitable platform for measuring glucose variation in environments with neutral pH. This is particularly relevant because the oxygen-containing functional groups on the CC surface increase the dehydrogenation rate of glucose oxidation in neutral phosphate-buffered saline.

MicroRNA ssc-miR-124a exhibits antiviral activity against porcine reproductive and respiratory syndrome virus via suppression of host genes CD163.

Porcine reproductive and respiratory syndrome (PRRS) is a serious infectious disease in the swine industry, which causes severe economic losses to current swine production worldwide. There are no effective antiviral strategies for preventing this disease. Previous studies showed that microRNAs (miRNAs) play important role in virus-host interactions. In this study, we demonstrated that the expression level of ssc-miR-124a was significantly downregulated during both high and low pathogenic PRRSV infection. Overexpression of ssc-miR-124a markedly inhibits PRRSV replication in PAMs. Luciferase reporter experiments and RISC immunoprecipitation assay were used to identify the ssc-miR-124a could directly target the 3'UTR of pig CD163 mRNA in a sequence-specific manner and that CD163 mRNA and protein levels were reduced in PAMs overexpressing ssc-miR-124a. These data not only provide new insights into virus-host interactions during PRRSV infection, but also suggest potential new antiviral strategies against PRRSV infection in the future.

Photocrosslinking silver nanoparticles-aloe vera-silk fibroin composite hydrogel for treatment of full-thickness cutaneous wounds.

Damage to the skin causes physiological and functional issues. The most effective treatment approach is the use of wound dressings. Silk fibroin (SF) is a promising candidate biomaterial for regulating wound healing; however, its antibacterial properties and biological activity must be further improved. In this study, a photocrosslinking hydrogel was developed to treat full-thickness cutaneous wounds. The composite hydrogel (Ag-AV-SF hydrogel) was prepared by introducing the silver nanoparticles (AgNPs) and aloe vera (AV) as the modifiers. In vitro study exhibited great antibacterial ability, biocompatibility and cell-proliferation and -migration-promoting capacities. It also showed the pH-response releasing properties which release more AgNPs in a simulated chronic infection environment. The healing effect evaluation in vivo showed the healing-promoting ability of the Ag-AV-SF hydrogel was stronger than the single-modifiers groups, and the healing rate of it reached 97.02% on Day 21, higher than the commercial wound dressing, silver sulfadiazine (SS) cream on sale. Additionally, the histological and protein expression results showed that the Ag-AV-SF hydrogel has a greater effect on the pro-healing regenerative phenotype with M2 macrophages at the early stage, reconstructing the blood vessels networks and inhibiting the formation of scars. In summary, the Ag-AV-SF hydrogel developed in this study had good physical properties, overwhelming antibacterial properties, satisfactory biocompatibility and significantly promoting effect on cell proliferation, migration and wound healing. Overall, our results suggest that the Ag-AV-SF hydrogel we developed has great potential for improving the wound healing in clinical treatment.

In-situ growth of 3D rosette-like copper nanoparticles on carbon cloth for enhanced sensing of ammonia based on copper electrodissolution.

Copper is an attractive candidate for sensing ammonia. Here, an electrodissolution mechanism for measuring liquid-phase ammonia was developed via a novel three-dimensional rosette-like structure of copper nanoparticles (CuNPs) integrated onto carbon cloth (CuNPs/CC). A one-step hydrothermal synthetic procedure was employed to construct the metallic CuNPs with a stereo rosette-like pattern on flexible CC substrate. The morphology, composition and sensing performance of the as-prepared composite were characterised in detail. The CuNPs/CC composite showed excellent sensing performance to ammonia, which is attributed to the electrodissolution of CuNPs being promoted by ammonia to form a stabilised copper-ammonia complex. This electrochemical response occurs without the electro-oxidation of ammonia, thus avoiding the energy barrier of the N-N bond and the toxicity of N-adsorbates, which is advantageous for ammonia detection. In addition, the sensor also shows very high sensitivity to ammonia with a low detection limit, as well as good anti-interference performance, repeatability and stability. The high accuracy and precision for the quantification of ammonia concentration in a variety of real samples indicate that the CuNPs/CC composition has potential in the development of high-performance ammonia sensors.

p13 from group II baculoviruses is a killing-associated gene.

p13 gene was first described in Leucania separata multinuclear polyhedrosis virus (Ls-p13) several years ago, but the function of P13 protein has not been experimentally investigated to date. In this article, we indicated that the expression of p13 from Heliothis armigera single nucleocapsid nucleopolyhedrovirus (Ha-p13) was regulated by both early and late promoter. Luciferase assay demonstrated that the activity of Ha-p13 promoter with hr4 enhancer was more than 100 times in heterologous Sf9 cells than that in nature host Hz-AM1 cells. Both Ls-P13 and Ha-P13 are transmembrane proteins. Confocal microscopic analysis showed that both mainly located in the cytoplasm membrane at 48 h. Results of RNA interference indicated that Ha-p13 was a killing-associated gene for host insects H. armigera. The AcMNPV acquired the mentioned killing activity and markedly accelerate the killing rate when expressing Ls-p13. In conclusion, p13 is a killing associated gene in both homologous and heterologous nucleopolyhedrovirus.