Baicalin is an inhibitor of subgroup J avian leukosis virus infection.

Avian leukosis virus subgroup J (ALV-J) can cause great economic losses to the poultry industry worldwide. Baicalin, one of the flavonoids present in S.baicalensis Georgi, has been shown to have antiviral activities. To investigate whether baicalin has antiviral effects on the infection of ALV-J in DF-1 cells, the cells were treated with baicalin at different time points. We found that baicalin could inhibit viral mRNA, protein levels and overall virus infection in a dose- and time-dependent manner using a variety of assays. Baicalin specifically targeted virus internalization and reduced the infectivity of ALV-J particles, but had no effect on the levels of major ALV-J receptor and virus binding to DF-1 cells. Collectively, these results suggest that baicalin might have potential to be developed as a novel antiviral agent for ALV-J infection.

Development and evaluation of an immunochromatographic strip for rapid detection of capsid protein antigen p27 of avian leukosis virus.

A rapid immunochromatographic strip for detecting capsid protein antigen p27 of avian leukosis virus was successfully developed based on two high-affinity monoclonal antibodies. The test strip could detect not only 600pg purified recombinant p27 protein but also quantified avian leukosis virus as low as 70 TCID50, which has comparative sensitivity to the commercial enzyme-linked immunosorbent assay (ELISA) kit. For the evaluation of this test strip, 1100 samples consisting of cloacal swabs, meconium collected from the earliest stool of one day old chicken and virus isolates were assessed both by the strip and by the commercial ELISA kit. The agreement between these two tests was 93.91%, 93.42% and 100%, respectively. The sensitivity and specificity of the strip were also calculated by using the ELISA kit as the standard. This immunochromatographic strip provides advantages of rapid and simple detection of capsid protein antigen p27 of avian leukosis virus, which could be applied as an on-site testing assay and used for control and eradication programs of avian leukosis disease.

Genistein inhibits the replication of avian leucosis virus subgroup J in DF-1 cells.

To investigate the antiviral effects of genistein on the replication of avian leukosis virus subgroup J (ALV-J) in DF-1 cells, the cells were treated with genistein at different time points and the antiviral effects were examined by using a variety of assays. We determined that genistein strongly inhibited viral gene expression and decreased the viral protein level in the cell supernatant and the cytoplasm without alerting virus receptor expression and viral attachment. We also observed that genistein was not found to interfere with virus entry, but significantly inhibited both viral gene transcriptions at 24h post infection and virus release, which indicate that genistein exerts its inhibitory effects on the late phase of ALV-J replicative cycle. These results demonstrate that genistein effectively block ALV-J replication by inhibiting virus transcription and release in DF-1 cells, which may be useful for therapeutic drug design.

Monoclonal antibodies directed against chicken ß2-microglobulin developed with a synthesized peptide.

We developed a panel of monoclonal antibodies (MAb) against chicken ß2-microglobulin (chß2M) by fusions between SP2/0 myeloma cells and spleen cells from mice immunized with a synthesized peptide corresponding to positions 91-119 of the COOH domain of chß2M. Two of them, 6E7 and 3D1, identified as IgG1/κ, could react with chß2M protein from avian macrophage HD11 cells and human 293T cells transfected with pcDNA3.1-chß2M in immunofluorescence assays. Only a 12 kDa protein band of chß2M could be detected in the HD11 and 293T/chß2M cell lysates by Western blot analysis. Chicken ß2M in serum and plasma could be found in Western blot by MAb 3D1. Moreover, MAb 3D1 also recognized the chß2M antigen on the cell membranes in flow cytometry. Immunohistochemical staining with these MAbs revealed that chß2M was present in chicken thymus, spleen, and bursa. These MAbs will be good tools for analyzing the mechanism of the chicken immune system.

[Development and characterization of monoclonal antibody against human FcR γ chain].

AIM: The current study was aimed to generate mouse monoclonal antibodies (mAbs) specific for Fc receptor common γ chain (FcRγ), which serves a critical signaling molecule for numerous human immune receptors that play essential roles in human immune responses. METHODS: Two BALB/c mice were immunized with the synthesized polypeptides containing 19 residues at the carboxyl terminus of human FcR γ chain. The spleen cells from the immunized mice were fused with myeloma Sp2/0 cells. ELISA was used to screen mAbs and the positive clones were selected for further characterization with Western blot and flow cytometry assays. RESULTS: After screen, there hybridoma cell lines which secreted mAbs specifically recognized the FcR γ chain were obtained and named as 5B6, 7D3 and 8D4. The 7D3 mAb had the best quality and it could recognize the FcR γ chain in vivo and in vitro with 2.5 µg/mL. CONCLUSION: We have generated the FcRγ-specific mAbs, which may serve as a valuable tool in the functional studies of FcRγ associated immune receptors.

[Differentiation of recombinant adenoviruses expressed MDV-1 VP22 subcellular localization pattern].

AIM: To study the transduction and localization mechanism of Marek's disease virus serotype 1 (MDV-1) CVI988 VP22 in different cells. METHODS: VP22 was expressed with recombinant adenovirus and identified by immunofluorescence assay (IFA) and Western blot. Lysates of recombinant virus infected 293 cells were added to normal MDBK cells to identify the transduction property of VP22. AD-293 cells infected with recombinant virus were fixed to investigate localization of VP22 at different time post infection. Transient expression of VP22 in AD-293 cells was carried out for control. RESULTS: The results showed that the VP22 expressed by recombinant adenovirus entered almost all the monolayer cells, which indicate the VP22 remains its transduction property. The VP22 first gather round the nucleus membrane, and then concentrated in particles in cytoplasm of 293 cells infected with recombinant adenovirus, compared with the nuclei localization pattern of VP22 in MDV infected CEF and transient expressed VP22 in 293 cells. CONCLUSION: The VP22 presented a different localization pattern in cells infected with different recombinant virus.

Discrimination of three medicinal materials from the Citrus genus by HPLC fingerprint coupled with two complementary software.

INTRODUCTION: Pericarpium Citri Reticulatae, Pericarpium Citri Reticulatae Viride and Fructus Aurantii come from the fruit of Citrus genus and possess analogous pharmacological activities. Either two or three of them are often present in one preparation. However, there is no standard method to differentiate the three herbal medicines for quality control. OBJECTIVE: To develop a fingerprint method for authentication of these three herbal medicines by HPLC. METHODOLOGY: Methanol extracts were analysed by HPLC, with a mobile phase of 0.1% phosphoric acid in water (A) and acetonitrile (B) in a gradient programme. The flow rate was set at 0.8 mL/min and UV detection at 320 nm. Principal component analysis and similarity evaluation were employed to analyse the chromatographic dataset. RESULTS: The chromatograms of 20 Pericarpium Citri Reticulatae, 13 Pericarpium Citri Reticulatae Viride and 15 Fructus Aurantii samples from diverse habitats had 13 peaks in common, and showed one peak characteristic for Pericarpium Citri Reticulatae Viride and two peaks characteristic for Fructus Aurantii. Furthermore, it was possible to differentiate the three medicines by a 'fingerprint region'. The difference between Pericarpium Citri Reticulatae and Fructus Aurantii was much bigger than that between Pericarpium Citri Reticulatae and Pericarpium Citri Reticulatae Viride, as could be shown by calculation of common peak ratio and variation peak ratio. CONCLUSION: A reliable HPLC fingerprint method coupled with principal component analysis and similarity evaluation was developed and showed substantial differentiation power for the three medicines.

[Construction and immunological characterization of recombinant Marek's disease virus expressing IBDV VP2 fusion protein].

A transfer plasmid vector pUC18-US10-VP2 was first constructed by inserting the gene of the enhancer green fluorescent protein(eGFP) fused to the VP2 gene of very virulent Infectious bursal disease virus (IBDV) JS strain into the US10 fragment of the Marek's disease virus (MDV) CV1988/Rispens. The recombinant virus, designated as rMDV, was developed by co-transfecting CEF with the transfer plasmid vector and simultaneously infecting with the CVI988/Rispens virus. The PCR and IFA results indicated that the rMDV is stable after 31 passages. Chickens vaccinated with rMDV were protected from challenge with 100LD50 of IBDV JS. The protection ratio of the chickens vaccinated with the 1000PFU, 2000PFU, 5000PFU of the rMDV were 50%, 60%, and 80% respectively. It is interesting that the average histopathology BF lesion scores of chicken group immunized with 5000PFU of rMDV by one-time vaccination was close to that of chicken group vaccinated with IBDV live vaccine NF8 strain for twice (2.0/1.5). There is no difference in protection between the groups (P > 0.05) but significent difference between groups immunized with 5000 PFU of rMDV and with normal MDV. This demonstrated that rMDV expressing VP2 fusion protein was effective vaccine against IBDV in SPF chickens.

[Experimental study on anti-pyretic effect of gegen qin lian decoction and its compounds].

OBJECTIVE: To investigate composition principles of Gegen Qin Lian decoction through anti-pyretic experiment. METHOD: Pharmacological effects of different compounds of Gegen Qin Lian decoction according to six hours temperature response index (TRI6) and average top temperature response height (deltaT) after the decoction was given to feverish animal model by inactived bacteria suspension. RESULT: As for reducing six hour temperature response index, Scutellaria baicalensis root was the main effective drug. Pueraria lobata root could enforce the effect while Coptis chinensis rhizome and Glycyrrhiza uralensis root counteracted it. As for reducing average top temperature response height, the Effects of four herbal drugs were the same as for TRI6. CONCLUSION: Of the compounds of Gegen Qin Lian decoction, as to the pharmcological anti-pyretic effects, the best one is the compound of Scutellaria baicalensis and Pueraria lobata roots.