Simultaneous Analysis of Hydrophobic Atractylenolides, Atractylon and Hydrophilic Sugars in Bai-Zhu Using a High-Performance Liquid Chromatography Column Tandem Technique.

An analytical method was established using high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors (HPLC-DAD-ELSD) with -C18 and -NH2 column tandem for the simultaneous determination of hydrophobic atractylenolide I, II, III, atractylone and hydrophilic compounds glucose, fructose and sucrose in the dried rhizome of Atractylodes macrocephala Koidz (a natural raw material for health foods, Bai-Zhu aka. in Chinese). The method combines the different separation capabilities of reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography. It can provides a new choice for the simultaneous determination of hydrophilic and hydrophobic compounds in traditional Chinese medicines and health foods. It provided a reference method for the quality control of Bai-Zhu. The results showed that the linear correlation coefficients of the established column tandem chromatographic method were all greater than 0.9990, the relative standard deviation was 0.1-2.8%, and the average recovery was 96.7-103.1%. The contents of atractylenolide I, II, III, atractylone, fructose, glucose, and sucrose in 17 batches of Baizhu were 172.3-759.8 µg/g, 201.4-612.8 µg/g, 160.3-534.2 µg/g, 541.4-8723.1 µg/g, 6.9-89.7 mg/g, 0.7-7.9 mg/g, and 1.2-21.0 mg/g, respectively.

Pilot Study of Robot-Assisted Teleultrasound Based on 5G Network: A New Feasible Strategy for Early Imaging Assessment During COVID-19 Pandemic.

Early diagnosis is critical for the prevention and control of the coronavirus disease 2019 (COVID-19). We attempted to apply a protocol using teleultrasound, which is supported by the 5G network, to explore the feasibility of solving the problem of early imaging assessment of COVID-19. Four male patients with confirmed or suspected COVID-19 were hospitalized in isolation wards in two different cities. Ultrasound specialists, located in two other different cities, carried out the robot-assisted teleultrasound and remote consultation in order to settle the problem of early cardiopulmonary evaluation. Lung ultrasound, brief echocardiography, and blood volume assessment were performed. Whenever difficulties of remote manipulation and diagnosis occurred, the alternative examination was repeated by a specialist from another city, and in sequence, remote consultation was conducted immediately to meet the consensus. The ultrasound specialists successfully completed the telerobotic ultrasound. Lung ultrasound indicated signs of pneumonia with varying degrees in all cases and mild pleural effusion in one case. No abnormalities of cardiac structure and function and blood volume were detected. Remote consultation on the issue of manipulation practice, and the diagnosis in one case was conducted. The cardiopulmonary information was delivered to the frontline clinicians immediately for further treatment. The practice of teleultrasound protocol makes early diagnosis and repeated assessment available in the isolation ward. Ultrasound specialists can be protected from infection, and personal protective equipment can be spared. Quality control can be ensured by remote consultations among doctors. This protocol is worth consideration as a feasible strategy for early imaging assessment in the COVID-19 pandemic.

[Pharmacokinetics of loganin, ferulic acid and stilbene glucoside in Bushen Tongluo formula in vivo].

To study the pharmacokinetics characteristic of loganin, ferulic acid and stilbene glucoside in rat plasma after oral administration of Bushen Tongluo formula. The plasma samples were treated by using liquid-liquid extraction technique, the concentrations were determined by HPLC-UV. Johnson spherigel C18 column (4.6 mm x 250 mm, 5 µm) was adopted and eluted with the of mobile phase of methanol-water containing 0.01% glacial acetic acid in a gradient mode, with the flow rate at 1.0 mL x min(-1), column temperature at 30 degrees C and injection volume of 10 µL. According to the findings, loganin was determined at 235 nm, ferulic acid and stilbene glucoside were determined at 320 nm, with the sample size of 10 µL. The pharmacokinetic parameters of loganin, ferulic acid and stilbene glucoside were calculated by DAS 2. 0 software as follows: C(max) was (0.369 ± 0.042), (0.387 ± 0.071), (0.233 ± 0.044) mg x L(-1); t(max) was (0.226 ± 0.022), (0.282 ± 0.031), (0.233 ± 0.044) h; t(½ß) was (6.89 ± 0.20), (10.73 ± 0.11), (6.93 ± 0.09) h; AUC(0-∞) was (1.91 ± 0.36), (3.22 ± 0.52), (1.52 ± 0.33) mg x h x L(-1); AUCO(0-t) was (1.62 ± 0.33), (2.58 ± 0.43), (1.30 ± 0.30) mg x h x L(-1); CL was (20.2 ± 4.0), (1.39 ± 0.23), (31.7 ± 6.9) L x h(-1) x kg(-1), respectively. The results showed that after the oral administration with Bushen Tongluo formula, loganin, ferulic acid and stilbene glucoside showed concentration-time curves in conformity with the two compartment model, with a rapid absorption, loganin and stilbene glucoside was excreted at a moderate speed, and ferulic acid was excreted slowly (but with the highest bioavailability). Bushen Tongluo formula can main maintain plasma concentration with three administrations everyday and so is suitable to be made into common oral preparation.

Enhanced Th1-biased immune efficacy of porcine circovirus type 2 Cap-protein-based subunit vaccine when coadministered with recombinant porcine IL-2 or GM-CSF in mice.

Porcine circovirus type 2 (PCV2) capsid (Cap) protein is the primary protective antigen responsible for inducing PCV2-specific protective immunity, so it is a desirable target for the development of recombinant subunit vaccines to prevent PCV2-associated diseases. Interleukin 2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF), used as immune adjuvants, have been shown to enhance the immunogenicity of certain antigens or vaccines in various experimental models. In this study, five different subunit vaccines (the PCV2-Cap, Cap-PoIL-2, PCV2-Cap + PoIL-2, Cap-PoGM-CSF, and PCV2-Cap + PoGM-CSF vaccines) were prepared based on baculovirus-expressed recombinant proteins. The immunogenicity of these vaccines was evaluated to identify the immunoenhancement by PoIL-2 and PoGM-CSF of the Cap-protein-based PCV2 subunit vaccine in mice. The PCV2-Cap + PoIL-2, Cap-PoGM-CSF, PCV2-Cap + PoGM-CSF, and PCV2-Cap vaccines induced significantly higher levels of PCV2-specific antibodies than the Cap-PoIL-2 vaccine, whereas there was no apparent difference between these four vaccines. Our results indicate that neither PoIL-2 nor PoGM-CSF had effect on the enhancement of the humoral immunity induced by the PCV2-Cap vaccine. Furthermore, the PCV2-Cap + PoIL-2, Cap-PoGM-CSF, and PCV2-Cap + PoGM-CSF vaccines elicited stronger lymphocyte proliferative responses and greater IL-2 and interferon gamma (IFN-γ) secretion. This suggests that PoIL-2 and PoGM-CSF substantially augmented the Th1-biased immune response to the PCV2-Cap vaccine. Following challenge, the viral loads in the lungs of the PCV2-Cap + PoIL-2-, Cap-PoGM-CSF-, and PCV2-Cap + PoGM-CSF-treated groups were dramatically lower than those in the Cap-PoIL-2- and PCV2-Cap-treated groups, indicating that the three vaccines induced stronger protective effects against challenge. These findings show that PoIL-2 and PoGM-CSF essentially enhanced the Th1-biased protective efficacy of the PCV2-Cap vaccine when coadministered with the protein or delivered as Cap-PoGM-CSF, and that the "antigen-cytokine"- or "antigen + cytokine"-based vaccines that we report here provide new basis for the development of safer and more effective vaccines.

A porcine circovirus type 2 (PCV2) mutant with 234 amino acids in capsid protein showed more virulence in vivo, compared with classical PCV2a/b strain.

BACKGROUND: Porcine circovirus type 2 (PCV2) is considered to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), which has become a serious economic problem for the swine industry worldwide. The major genotypes, PCV2a and PCV2b, are highly prevalent in the pig population and are present worldwide. However, another newly emerging PCV2b genotype mutant, which has a mutation in its ORF2-encoded capsid protein, has been sporadically present in China, as well as in other countries. It is therefore important to determine the relative virulence of the newly emerging PCV2b genotype mutant, compared with the existing PCV2a and PCV2b genotypes, and to investigate whether the newly emerging mutant virus induces more severe illness. METHODOLOGY/PRINCIPAL FINDINGS: Twenty healthy, 30-day-old, commercial piglets served as controls or were challenged with PCV2a, PCV2b and the newly emerging mutant virus. A series of indexes representing different parameters were adopted to evaluate virulence, including clinical signs, serological detection, viral load and distribution, changes in immune cell subsets in the peripheral blood, and evaluation of pathological lesions. The newly emerging PCV2 mutant demonstrated more severe signs compatible with PMWS, characterized by wasting, coughing, dyspnea, diarrhea, rough hair-coat and depression. Moreover, the pathological lesions and viremia, as well as the viral loads in lymph nodes, tonsils and spleen, were significantly more severe (P<0.05) for piglets challenged with the newly emerging mutant compared with those in the groups challenged with PCV2a and PCV2b. In addition, a significantly lower average daily weight gain (P<0.05) was recorded in the group challenged with the newly emerging PCV2 mutant than in the groups challenged with the prevailing PCV2a and PCV2b. CONCLUSIONS: This is believed to be the first report to confirm the enhanced virulence of the newly emerging PCV2 mutant in vivo.

Identification of three new type-specific antigen epitopes in the capsid protein of porcine circovirus type 1.

Porcine circovirus type 1 (PCV1) has been identified as a contaminant of porcine kidney cell line (PK-15). Serological evidence and genetic studies have suggested that PCV1 is widespread in domestic pigs. In this study, monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were generated against a recombinant PCV1 Cap protein (PCV1-Cap), which was expressed using the baculovirus system. PEPSCAN analysis was used to identify epitopes on the PCV1-Cap with mAbs and pAbs. Three linear B-cell epitopes, including residues (85)GGTNPLP(91), (162)FTPKPELDKTIDWFHPNNK(180) and (219)YVQFREFILKDPLNK(233), specific for PCV1-Cap, were finely defined. These results will facilitate future investigations into antigenic differences and differential diagnosis between PCV1 and PCV2.

Porcine circovirus genotype 2a (PCV2a) and genotype 2b (PCV2b) recombinant mutants showed significantly enhanced viral replication and altered antigenicity in vitro.

Two recombinant mutants of porcine circovirus type 2 (PCV2), which resulted from replacement of a genomic fragment containing the open reading frame 2 (ORF2) of genotype PCV2b with that of genotype PCV2a, were obtained initially from co-infection with PCV2a and 2b genotype viruses in vitro. The two mutant viruses contained the ORF1 sequence from genotype PCV2b and the ORF2 sequence from genotype PCV2a. They were designated according to the nomenclature proposed by Grau et al., indicating the origin of the ORF1 sequence first and that of the ORF2 sequence second, i.e., PCV2b(JF11)/2a(CL1) and PCV2b(YJ)/2a(CL1). The replication efficiencies of the two PCV2 recombinant mutants were enhanced significantly and their antigenicities were altered significantly in vitro when compared with their parental strains.

Construction and biological characterisation of recombinant porcine circovirus type 2 expressing the V5 epitope tag.

Porcine circovirus type 2 (PCV2) is a major causal agent of post-weaning multisystemic wasting syndrome in piglets. To investigate the feasibility of PCV2 expressing an exogenous epitope, a 14-amino-acid V5 epitope derived from simian parainfluenza virus type 5, was inserted into the C terminus of the capsid protein. Recombinant PCV2 expressing the V5 epitope, recPCV2/CL-V5, was rescued by transfecting an infectious clone into PK-15 cells and was characterised by an immunoperoxidase monolayer assay (IPMA), a serum neutralisation assay (SNA), a capture enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy. The V5 epitope was detected in the recombinant marker virus by IPMA and capture ELISA. Furthermore, there was no detectable difference in the antigenicity of the recombinant marker virus compared with the parental virus by IPMA and SNA using PCV2-positive serum and the neutralising monoclonal antibody 1D2. However, recPCV2/CL-V5 marker virus could be differentiated from the parental virus by PCR, IPMA and capture ELISA. The recombinant marker virus was stable on multiplication through 10 passages in PK-15 cells, with a maximum titre of 10(6.25) 50% tissue culture infective dose (TCID(50))/ml. BALB/c mice were inoculated with the recombinant or parental virus via the intranasal and intraperitoneal routes. The parental and recombinant viruses both could replicate in mice, cause microscopic pathological changes, and induce mice to generate anti-PCV2 antibodies. Furthermore, the recombinant marker virus could also induce anti-V5 epitope tag antibodies. These results indicated that V5 epitope could be displayed on the surface of the capsid protein by inserting its gene just before stop codon of open reading frame 2. More importantly, insertion of the V5 epitope did not seem to interfere with biological characterisation of the recPCV2/CL-V5 marker virus.

Development of a blocking ELISA for detection of serum neutralizing antibodies against porcine circovirus type 2.

A monoclonal antibody (Mab)-based blocking ELISA was developed for the detection of serum neutralizing antibodies to porcine circovirus type 2 (PCV2). The Mab with neutralizing activity, which was produced by immunizing a recombinant capsid protein of PCV2 expressed in insect cells, was used as the detector antibody. The assay was evaluated in comparison with a serum neutralization assay, and its sensitivity and specificity were determined to be 98.8% and 88.5%, respectively. A significant positive correlation was found between results of the blocking ELISA and the serum neutralization assay (r=0.9381). The assay was verified by testing experimental and commercial pig sera. A longitudinal antibody profile showed that serum neutralizing antibodies were detected 2 weeks after vaccination and that the detection rate reached 100% at 4 weeks. The serum neutralizing antibody profile showed a decrease from the age of 4 to 13 weeks, and seroconversion after 13 weeks in pigs from a commercial pig farm. Additionally, the positive detection rate in 703 sera collected from nine commercial pig farms was 73%. This report demonstrates that the assay is a simple, specific, sensitive and convenient method for epidemiological surveys and evaluations of serum neutralizing antibodies against PCV2.

Identification of a new antigen epitope in the nuclear localization signal region of porcine circovirus type 2 capsid protein.

OBJECTIVE: Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic syndrome in pigs. The capsid (Cap) protein encoded by ORF2 is the main structural protein involved in the host immune protective response to PCV2. It is therefore important to map the antigenic epitopes of the PCV2 Cap protein. METHODS: In this study, 5 monoclonal antibodies (mAbs) against the recombinant PCV2 Cap protein, expressed by the baculovirus system in Sf21 insect cells, were generated. The antigenic epitope recognized by these mAbs was located in the Cap A protein by Western blot analysis using 4 overlapping minifragments covering the Cap protein expressed in Escherichia coli. To locate the epitope more accurately, 3 sets of overlapping peptides were synthesized. RESULTS: The results demonstrated that 4 of the 5 mAbs recognized the same core epitope ((26)RPWLVHPRHRY(36)) located in the nuclear localization signal (NLS) region at the N terminus of the Cap protein. The other mAb (1D2) reacted with the recombinant Cap protein only, indicating that it recognizes a potential conformational epitope. This mAb demonstrated a neutralizing effect on PCV2. CONCLUSION: This is the first study to identify an antigenic epitope in the NLS region of the PCV2 Cap protein using mAbs. The results of this study will facilitate future investigations into the mechanism and function of nuclear localization of this protein.