To construct the plasmid of BAC-HSV-1 with GFP reporter gene and research the biological property of its infectious progeny virus. We constructed the plasmid C223-UL43-left-arms-UL47-right-arms which carried the homologous sequences of HSV-1. Liposome embedding method was used to transfect HSV-1 genome and the plasmid C223-UL43-left-arms-UL47-right-arms linearized by Mlu I digestion into Vero cells. After the successful homologous recombination in the eukaryotic cells, the recombinant BAC-HSV-1 with GFP reporter gene was generated. Then, the positive CPE were taken by plaque purification and by hirt extraction during the moment of the circularization of HSV-1 DNA, and the plasmid of BAC -HSV-1 was acquired. Electroporation was used to transfect the BAC -HSV-1 into DH10B, and then the single colonies of interest were confirmed both by MluI digestion and PCR. Experimental group and the control group cells were given BAC-HSV-1 plasmid and HSV-1 genomic DNA respectively to produce the BAC-HSV-1 and HSV-1 progeny virions. Vero cells were inoculated with the progeny virions at MOI = 0.1 and then a TCID50 assay was performed to determine the titers of virons in the two groups at 48 hours post inoculation. The plasmid BAC-HSV-1 was successfully constructed by the restriction enzyme analysis and the PCR. The titers of progeny virions were calculated by the TCID50 assay. No significant difference in the titers of virions between two groups was observed (P > 0.05). The infectious BAC-HSV-1 shuttle virus/plasmid between eukaryotic and prokaryotic cells was successfully constructed.
Animals ; Cercopithecus aethiops ; Chromosomes, Artificial, Bacterial ; Green Fluorescent Proteins ; genetics ; Herpesvirus 1, Human ; genetics ; pathogenicity ; Recombination, Genetic ; Vero Cells

OBJECTIVETo establish an animal model of Bell palsy induced by type I herpes simplex virus (HSV-1) infection and to assess the role and site of HSV-1 in the pathogenesis of facial paralysis.

METHODSFifty-three female Balb/c mice four-week-old weighted 16-18 g were studied. After scratching the surface of bilateral auricles with a 26-gauge needle, 25 microL HSV-1 with a titer of 6. 7 x 10(7) PFU/ml was inoculated into the right auricle, and the same volume of PBS was placed in the left. As a control, PBS was placed on the bilateral auricles of 4 mice. The HSV-1 DNA in bilateral facial nerve, bilateral brainstem, bilateral trigeminal carrier ganglion, bilateral brain, and blood at different stage was examined with polymerase chain reaction analysis.

RESULTSThirty-seven animals (75.51%) appeared different degree facial paralysis among the 49 inoculated animals. Fourteen facial paralysis (37.84%) were on the right, 3 (8.11%) on the left, and 20 (54.05%) on the bilateral side. Six animals with facial palsy were recovered during 3-13 days, the average recovery time was 7.83 days.

CONCLUSIONSThe existence of HSV-1 in the brainstem and the cerebral cortex is significant for facial paralysis.

Animals ; Bell Palsy ; virology ; Disease Models, Animal ; Female ; Herpes Simplex ; physiopathology ; Herpesvirus 1, Human ; pathogenicity ; Mice ; Mice, Inbred BALB C ; Motor Cortex ; virology

OBJECTIVETo establish an animal model of Bell's palsy induced by the reactivation of latent herpes simplex virus type 1 (HSV-1), and observe the effect of interferon and IgG on the facial nerve paralysis induced by HSV-1 infection. METHODS Totally 64 four-week-old female Balb/c mice weighted 16-18 gram were selected. Using scratching the surface of bilateral auricles by a 26-gauge needle, 25 microl HSV-1 with a titer of 6.7 x 10(8) PFU/ml was inoculated into the left auricle and the same volume of PBS was placed in the right in order to develop a mouse model of latent HSV-1. In this study, interferon and IgG administration were performed before and after facial nerve paralysis and continued for 3 days. Controls were given normal sodium instead of interferon and IgG, and the incidence and duration of facial nerve paralysis were compared in the groups interferon and IgG and control. Ciclosporin was given to the mice eight weeks after recovery from facial nerve paralysis caused by inoculation with HSV-1. The HSV-1 DNA in bilateral facial nerve and bilateral trigeminal ganglion after the treatment were examined with polymerase chain reaction (PCR) analysis. RESULTS There were 10 mice of facial nerve paralysis in the first group. The incidence of facial nerve paralysis was 50% and the duration of facial nerve paralysis was (7.2 +/- 2.2) days. There were 6 mice of facial nerve paralysis in the second group. The incidence of facial nerve paralysis was 30% and the duration of facial nerve paralysis was (4.5 +/- 1.8) days. There were 16 mice of facial nerve paralysis in the control group. The incidence of facial nerve paralysis was 67% and the duration of facial nerve paralysis was (8.9 +/-2.6) days. IgG didn't reduce the incidence and duration of facial nerve paralysis by statistics analysis (P > 0.05), but interferon reduced the incidence and duration of facial nerve paralysis (P < 0.05). After administration of ciclosporin, 3/28 of mice developed facial nerve paralysis. The HSV-1 DNA was detected from facial nerve of all the mice of facial palsy. No facial palsy was observed in mice in which no HSV-1 DNA was detected from facial nerve.

CONCLUSIONSFacial nerve paralysis might be caused by reactivation of latent HSV-1, and the reactivation might be related with immunosuppression. Administration of interferon reduces the incidence and duration of facial nerve paralysis. Administration of IgG can't reduced the incidence and duration of facial nerve paralysis.

Animals ; DNA, Viral ; isolation & purification ; Disease Models, Animal ; Facial Paralysis ; etiology ; prevention & control ; virology ; Female ; Herpes Simplex ; complications ; pathology ; Herpesvirus 1, Human ; pathogenicity ; Immunoglobulin G ; therapeutic use ; Interferons ; therapeutic use ; Mice ; Mice, Inbred BALB C ; Recurrence

OBJECTIVETo observe the effect of the extract from gardenia on influenza viral pneumonia in mice and virus-induced cytopathic effect.

METHODThe mice were infected by influenza virus in nasal, the lung inflammation, mortality rate and life elongation rate were observed respectively. The anti-viral activity of the extract from gardenia was accessed by cytopathic effect (CPE) in vitro and 0% toxicity concentration (TC0), 50% toxicity concentration (TC50), 50% inhibitor concentration (IC50), therapeutic index (TI) were determined by Reed-Muench method.

RESULTThe pneumonia induced by influenza virus in mice was inhibited significantly by the extract from gardenia, as the mortality rate decreased and the life elongation rate increased remarkably. Meanwhile the NO content in serum decreased significantly; The cytopathic effect induced by six kinds of viruses was inhibited remarkably.

CONCLUSIONThe six kinds of viruses were inhibited significantly by the extract from gardenia which inhibitory effect on mice influenza viral pneumonia was related to the NO content decreased.

Animals ; Antiviral Agents ; pharmacology ; Cells, Cultured ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Epithelial Cells ; cytology ; virology ; Esophagus ; cytology ; virology ; Female ; Gardenia ; chemistry ; Herpesvirus 1, Human ; drug effects ; Humans ; Influenza A Virus, H1N1 Subtype ; drug effects ; Male ; Mice ; Nitric Oxide ; blood ; Orthomyxoviridae ; pathogenicity ; Plants, Medicinal ; chemistry ; Pneumonia, Viral ; blood ; drug therapy ; Random Allocation ; Respiratory Syncytial Virus, Human ; drug effects

RT-PCR was used to detect expression level of VP16 mRNA and IFN-gamma mRNA in Herpes simplex virus type-1 infected mice brains at 4th day, 7th day, 10th day, 14th day, 21st day post infection and investigate the effects of the Gardenia extracts-T9 on viral replication and host immunity. The results showed that expression of VP16 mRNA in Gardenia extracts-T9 high dose and low dose group were both lower than that in virus control group at same time point. Relative VP16 mRNA expression in low dose group decreased at 21st day and relative VP16 mRNA expression in high dose group decreased continuously. Relative expression of IFN-gamma mRNA in high dose and low dose groups were both higher than that in virus control group at all time point except the 4th day. IFN-gamma mRNA in low dose group increased from the 4th day till the 14th day, and after the 14th day, the expression decreased slightly. Relative IFN-gamma mRNA in high dose group maintained increasing from 4th day till 21st day. Base on these results, we conclude that Gardenia extracts-T9 might exert the inhibition effect of viral replication by upregulating expression of IFN-gamma mRNA.
Animals ; Brain ; metabolism ; virology ; Female ; Gardenia ; chemistry ; Gene Expression Regulation, Viral ; drug effects ; genetics ; Herpesvirus 1, Human ; drug effects ; growth & development ; pathogenicity ; Interferon-gamma ; genetics ; Mice ; Mice, Inbred BALB C ; Plant Extracts ; administration & dosage ; pharmacology ; Polymerase Chain Reaction ; RNA, Messenger ; genetics ; Virus Replication ; drug effects

OBJECTIVETo investigate the immune responses and protection from virus challenge, induced by the coinjection of IL-2cDNA with herpes simplex virus type 1 (HSV-1) glycoprotein-D (gD) DNA vaccine.

METHODSTwo DNA vaccines (pgD and pIL-2) were constructed by inserting the gD gene and IL-2 cDNA into the eukaryotic expression vector pcDNA3.1, respectively. The BALB/c mice were inoculated intramuscularly three times at 2-week intervals. Two weeks after the final immunization, mice were bled for antibody assay and spleen cells were separated for Th cell proliferation and cytokine assays. Delayed type hypersensitivity (DTH) response was detected by the pinna-swelling test. Corneal protection under HSV-1 virus challenge was continuously observed with slit-lamp microscope.

RESULTSIL-2 cDNA coinjection remarkably enhanced the specific IgG2a level when compared with gD plasmid vaccination alone. Th cell proliferation and secretion of cytokines (IL-2 and IFN-gamma) were significantly increased by IL-2 cDNA coinjection. However, the production of IL-10 was inhibited. The DTH response was also enhanced by IL-2 coinjection. When the mice were challenged with HSV-1, the cornea epithelial lesions were significantly alleviated by IL-2 coinjection as compared with gD vaccination alone.

CONCLUSIONIL-2 cDNA can enhance both the humoral and cellular immune responses, and thus increase the vaccine potency.

Animals ; Antibodies, Viral ; blood ; COS Cells ; Cell Proliferation ; Cercopithecus aethiops ; DNA ; genetics ; Female ; Herpesvirus 1, Human ; pathogenicity ; Hypersensitivity, Delayed ; immunology ; Immunization ; Immunoglobulin G ; blood ; Interferon-gamma ; blood ; Interleukin-2 ; biosynthesis ; genetics ; Mice ; Mice, Inbred BALB C ; Random Allocation ; Th1 Cells ; cytology ; Transfection ; Vaccines, DNA ; immunology ; Viral Envelope Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

GITR (glucocorticoid-induced TNF receptor) is a recently identified member of the TNF receptor superfamily. The receptor is preferentially expressed on CD4+CD25+ regulatory T cells and GITR signals break the suppressive activity of the subset. In this study, we wanted to reveal the in vivo function of GITR in herpes simplex virus type 1 (HSV-1) infection. A single injection of anti-GITR mAb (DTA-1) immediately after viral infection significantly increased the number of CD4+ and CD8+ T cells expressing CD25, an activation surface marker, and secreting IFN-gamma. We confirmed these in vivo observations by showing ex vivo that re-stimulation of CD4+ or CD8+ T cells with a CD4+ or CD8+ T-cell-specific HSV-1 peptide, respectively, induced a significant elevation in cell proliferation and in IFN-gamma secretion. Our results indicate that GITR signals play a critical role in the T-cell immunity to HSV-1.
Animals ; Antibodies, Monoclonal/pharmacology ; CD4-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; Cell Proliferation ; Female ; Glucocorticoids/*pharmacology ; Herpes Simplex/*immunology ; Herpesvirus 1, Human/pathogenicity ; *Immunity, Cellular ; Interferon Type II/secretion ; *Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Peptide Fragments/metabolism ; Receptors, Interleukin-2/metabolism ; Receptors, Nerve Growth Factor/genetics/immunology/*metabolism ; Receptors, Tumor Necrosis Factor/genetics/immunology/*metabolism ; Research Support, Non-U.S. Gov't ; T-Lymphocytes/*immunology/metabolism/virology