聚肌胞联合维生素C微量激素治疗单疱性角膜炎

聚肌胞联合维生素Ｃ微量激素治疗单疱性角膜炎福建泉州市国专医院眼科姚照华单抱病毒性角膜炎易复发，致盲率高。尽管近年来本病的治疗有了很大的进展，但尚缺乏理想的疗效。作者自ｌｏｐ年始用干扰素诱导剂聚肌胞联合维生素Ｃ微量地塞米松结膜下注射治疗单癌病毒性角膜炎...

Effect of HSV-2 Infected Monocytes on the Production of TNF-a and IL-6

Objective: In order to detect the role of monocytes inHSV-2 infection, we studied the effect of herpes sim-plex Virus-2 infection on the production of tumor ne-crosis factor (TNF-σ), interleukin-6 (IL-6) secretedby monocytes. Methods: Monocytes were infected by HSV-2 (333Strain). Culture supernatants were collected at 1, 3,5, 7 days post-infection. The levels of TNF-α, IL-6were measured by enzyme-linked immunosorbent as-say (ELISA). Results: The levels of TNF-α secretion by mono-cytes significantly decreased on first day post-infection. The levels of IL-6 significantly decreasedon first and third days post-infection, and then gradu-ally increased to the control on seventh day post-infection. Conclusions: TNF-α and IL-6 production by mono-cytes was inhibited during HSV-2 infection. The pro-duction of cytokines may play an important role inherpes simplex viurs-2 pathogenicity and immunity.

Effect of Herpes Simplex Virus-2 Infection in Vitro on the Expression of HLA Class II Antigen of Monocytes

Objectives: In order to investigate the role of mono-cytes and human leukocyte antigen (HLA) class II an-tigen in herpes simplex virus-2 (HSV-2) infection, westudied the effect of HSV-2 infection in vitro on theexpression of HLA class II antigen on monocytes.Methods: Monocytes were infected with HSV-2(Strain 333). Culture cells were collected 1, 3, 5 and 7days after infection. The levels of expression of HLAclass II antigen were measured by using alkaline phos-phatase antialkaline phosphatase method (APAAP).Results: The levels of the expression of HLA class IIantigen on monocytes significantly decreased on thefirst day of post-infection, and then gradually returnedto levels found in the controls by the 7th day post-infection.Conclusion: HLA class II antigen expression onmonocytes was inhibited with HSV-2 infection, whichmight be one means of virus escape at an early phase.The expression of HLA class II antigen may play animportant role in herpes simplex viurs-2 pathogenic-ity and immunity.

The Herpes Simplex Virus Type 1 Multiple Function Protein ICP27

The herpes simplex virus type 1 （HSV-1） infected-cell protein 27 （ICP27） is an essential, highly conserved protein involved in various steps of HSV-1 gene regulation as well as in the shut-off of host gene expression during infection. It functions primarily at the post-transcriptional level in inhibiting precursor mRNA splicing and in promoting nuclear export of viral transcripts. Recently, many novel functions performed by the HSV- 1 ICP27 protein were shown, including leptomycin B resistance, inhibition of the type I interferon signaling, regulation of the viral mRNA translation and determining the composition of HSV-1 virions

In vitro Anti-viral Activity of the Total Alkaloids from Tripterygium hypoglaucum against Herpes Simplex Virus Type 1

Herpes simplex virus type 1 （HSV-1） is a commonly occurring human pathogen worldwide. There is an urgent need to discover and develop new alternative agents for the management of HSV-1 infection. Tripterygium hypoglaucum （level） Hutch （Celastraceae） is a traditional Chinese medicine plant with many pharmacological activities such as anti-inflammation, anti-tumor and antifertility. The usual medicinal part is the roots which contain about a 1% yield of alkaloids. A crude total alkaloids extract was prepared from the roots of T. hypoglaucum amd its antiviral activity against HSV-1 in Vero cells was evaluated by cytopathic effect （CPE） assay, plaque reduction assay and by RT-PCR analysis. The alkaloids extract presented low cytotoxicity （CC50 = 46.6μg/mL） and potent CPE inhibition activity, the 50% inhibitory concentration （ICs0） was 6.5 μg/mL, noticeably lower than that of Acyclovir （15.4μg /mL）. Plaque formation was significantly reduced by the alkaloids extract at concentrations of 6.25 μg/mL to 12.5 μg/mL, the plaque reduction ratio reached 55% to 75% which was 35% higher than that of Acyclovir at the same concentration. RT-PCR analysis showed that, the transcription of two important delayed early genes UL30 and UL39, and a late gene US6 of HSV-1 genome all were suppressed by the alkaloids extract, the expression inhibiting efficacy compared to the control was 74.6% （UL30）, 70.9% （UL39） and 62.6% （US6） respectively at the working concentration of 12.5μg/mL. The above results suggest a potent anti-HSV-1 activity of the alkaloids extract in vitro.

Serial imaging of human embryonic stem-cell engraftment and teratoma formation in live mouse models

Two new types of lentiviral vectors expressing a reporter transgene encoding either firefly luciferase （fLuc） for bioluminescence imaging or the HSV1 thymidine kinase （HSV1-TK） for radiopharmaceutical-based imaging were constructed to monitor human embryonic stem cell （hESC） engraftment and proliferation in live mice after trans- plantation. The constitutive expression of either transgene did not alter the properties of hESCs in the culture. We next monitored the formation of teratomas in SCID mice to test （1） whether the gene-modified hESCs maintain their developmental pluripotency, and （2） whether sustained reporter gene expression allows noninvasive, whole-body imaging of hESC derivatives in a live mouse model. We observed teratoma formation from both types of gene-modified cells as well as wild-type hESCs 2-4 months after inoculation. Using an optical imaging system, bioluminescence from the fLuc-transduced hESCs was easily detected in mice bearing teratomas long before palpable tumors could be detected. To develop a noninvasive imaging method more readily translatable to the clinic, we also utilized HSV1-TK and its specific substrate, 1-（2＇-deoxy-2＇-fluoro-β-D-arabinofuranosyl）-5-[^125I]iodouracil（[^125I]FIAU）, as a reporter/ probe pair. After systemic administration, [^125I]FIAU is phosphorylated only by the transgene-encoded HSV1-TK enzyme and retained within transduced （and transplanted） cells, allowing sensitive and quantitative imaging by single-photon emission computed tomography. Noninvasive imaging methods such as these may enable us to monitor the presence and distribution of transplanted human stem cells repetitively within live recipients over a long term through the expression of a reporter gene.

Fhyroid hormone controls the gene expression of HSV-1 .AT and ICPO in neuronal cells

Various factors/pathways including hormonal regulation have been suggested to control herpes simplex virus type 1 （HSV-1） latency and reactivation. Our computer analysis identified a DNA repeat containing thyroid hormoneresponsive elements （TRE） in the regulatory region of HSV-1 latency-associated transcript （LAT）. Thyroid hormone （triiodothyronine, T3） functions via its receptor TR （thyroid hormone receptor）, a transcription factor. Present study investigated the roles of TR and T3 in HSV-1 gene expression using cultured neuoroblastoma cell lines. We demonstrated that liganded TR activated LAT transcription, but repressed infected cell protein no. 0 （ICP0） transcription in the presence of LAT TRE. Chromatin immunoprecipitation （CHIP） assays showed that TRs were recruited to LAT TREs independently of T3 and hyperacetylated H4 was associated with the LAT promoter that was transcriptionally active. In addition, ChIP results showed that the chromatin insulator protein CCCTC-binding factor was enriched at the LAT TREs in the presence of TR and T3. In addition, the BRG1 chromatin remodeling complex is found to participate in the T3/TR-mediated LAT activation since overexpression of BRG1 enhanced the LAT transcription and the dominant-negative mutant K785R abolished the activation. This is the first report revealing that TR elicits epigenetic regulation on HSV-1 ICP0 expression in neuronal cells and could have a role in the complex processes of HSV-1 latency/reactivation.

Feasibility of herpes simplex virus type 1 mutants labeled with radionuclides for tumor treatment

For over one hundred years, viruses have been recognized as capable of killing tumor cells. At present, people are still researching and constructing more suitable oncolytic viruses for treating different malignant tumors. Although extensive studies have demonstrated that herpes simplex virus type 1 (HSV-1) is the most potential oncolytic virus, therapies based on herpes simplex virus type 1 vectors still arouse bio-safety and risk management issues. Researchers have therefore introduced the new idea of treating cancer with HSV-1 mutants labeled with radionuclides, combining radionuclide and oncolytic virus therapies. This overview briefly summarizes the status and mechanisms by which oncolytic viruses kill tumor cells, discusses the application of HSV-1 and HSV-1 derived vectors for tumor therapy, and demonstrates the feasibility and prospect of HSV-1 mutants labeled with radionuclides for treating tumors.

The miRNAs of Herpes Simplex Virus(HSV)

Herpes simplex virus (HSV) is a group of common human pathogens with two serotypes HSV-1 and HSV-2.The prevalence of HSV is worldwide.It primarily infects humans through epithelial cells,when it introduces a latent infection into the nervous system.During viral latency,only a region known as the latency-associated transcript (LAT) is expressed.The discovery of HSV miRNAs helps to draw a larger picture of the infection and pathogenesis of the virus.This review summarizes miRNAs found in HSV-1 and HSV-2 so far.The functional studies of miRNAs in HSV to date indicate that they play a stage-specific role coordinated with viral proteins to maintain the virus life cycle.

Retrovirus-mediated herpes simplex virus thymidine kinase gene therapy approach for hepatocellular carcinoma

The therapeutic effect of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system on hepatocellular carcinoma was studied in this experiment. The tk-containing retroviral recombinants were used to infect hepatoma cells (BEL-7402) and the cells were treated with ganciclovir (0-1000 microg/ml). The results showed that HSV-tk gene could be efficiently transferred in vitro into hepatoma cells and stably expressed. The growth potential of the tk-containing cells was significantly inhibited by GCV (P

Antiviral Activity of Recombinant Cyanovirin-N against HSV-1

In this study,a standard strain of HSV-1 (strain SM44) was used to investigate the antiviral activity of the recombinant Cyanovirin-N (CV-N) against Herpes simplex virus type 1 (HSV-1) in vitro and in vivo.Cytopathic effect (CPE) and MTT assays were used to evaluate the effect of CV-N on HSV-1 in Vero cells.The number of copies of HSV-DNA was detected by real-time fluorescence quantitative PCR (FQ-PCR).The results showed that CV-N had a low cytotoxicity on Vero cells with a CC50 of 359.03±0.56 μg/mL,and that it could not directly inactivate HSV-1 infectivity.CV-N not only reduced the CPE of HSV-1 when added before or after viral infection,with a 50% inhibitory concentration (IC50) with 2.26 and 30.16μg/mL respectively,but it also decreased the copies of HSV-1 DNA in infected host cells.The encephalitis model for HSV-1 infection was conducted in Kunming mice,and treated with three dosages of CV-N (0.5,5 & 10 mg/kg) which was administered intraperitoneally at 2h,3d,5d,7d post infection.The duration for the appearance of symptoms of encephalitis and the survival days were recorded and brain tissue samples were obtained for pathological examination (HE staining).Compared with the untreated control group,in the 5mg/kg CV-N and 10mg/kg CV-N treated groups,the mice suffered light symptoms and the number of survival days were more than 9d and 14d respectively.HE staining also showed that in 5mg/kg CV-N and 10mg/kg CV-N treated groups,the brain cells did not show visible changes,except for a slight inflammation.Our results demonstrated that CV-N has pronounced antiviral activity against HSV-1 both in vitro and in vivo,and it would be a promising new candidate for anti-HSV therapeutics.

The Nucleolus and Viral Infection

The nucleolus is a subnuclear structure of eukaryocytes. It was thought that nucleolus only participates in the biogenesis and processing of rRNA. However, more and more evidence shows that it has many other functions, such as tRNA precursor processing, stress sensing and it is also involved in gene silencing, senescence and cell cycle regulation. Here, we summarize the recent understandings about the nucleolar functions, the regulation of nucleolar localization of proteins and the role that the nucleolus plays in virus infection, in which some related studies of Herpes simplex virus type 1 (HSV-1) US11, UL24 and bovine herpesvirus-1 infected cell protein 27 (BICP27) carried out in our lab will also be included.

Effect of siRNAs on HSV-1 Plaque Formation and Relative Expression Levels of RR mRNA

RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1 (HSV-1) RR is composed of two distinct homodimeric subunits encoded by UL39 and UL40,respectively. In this study,we applied siRNAs targeting the UL39 and UL40 genes of HSV-1. We showed that synthetic siRNA silenced effectively and specifically UL39 and UL40 mRNA expression and inhibited HSV-1 replication. Our work offers new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication.

Preparation of BFV Gag Antiserum and Preliminary Study on Cellular Distribution of BFV

Viruses (e.g. Human immunodeficiency virus, Human simplex virus and Prototype foamy virus) are obligate intracellular parasites and therefore depend on the cellular machinery for cellular trafficking. Bovine foamy virus (BFV) is a member of the Spumaretrovirinae subfamily of Retroviruses, however, details of its cellular trafficking remain unknown. In this study, we cloned the BFV gag gene into prokaryotic expression vector pET28a and purified the denaturalized Gag protein. The protein was used to immunize BALB/c mouse to produce antiserum, which could specifically recognize the BFV Gag protein in BFV-infected cells through western blot assay. Additionally, these results demonstrated that both the optimal and suboptimal cleavage of Gag protein occur in BFV-infected cells. Subsequently, the Gag antiserum was used to investigate subcellular localization of BFV. In immunofluorescence microscopy assays, colocalization microtubules (MTs) and assembling viral particles were clearly observed, which implied that BFV may transport along cellular MTs in host cells. Furthermore, MTs-depolymerizing assay indicated MTs were required for the efficient replication of BFV. In conclusion, our study suggests that BFV has evolved the mechanism to hijack the cellular cytoskeleton for its replication.

Current Status of Natural Products from Plants as Anti-herpes Simplex Virus 1 Agents

Nucleoside analogues have been the mainstay of clinical treatment of herpes simplex virus 1 (HSV-1) infections since their development. However, the emergence of drug resistant strains has underlined the urgency of the discovery of novel anti-HSV-1 drugs. Natural products, which provided many novel drug leads, are known to be an important source of anti-HSV-1 agents. Herein, we present an overview of natural products with anti-HSV-1 activities isolated from a variety of plants reported in recent years. Several different compounds, mainly belonging to the three groups of polysaccharides, polyphenols and terpenes, showed antiviral effects against HSV-1, indicating their potential to be promising anti-HSV-1 agents.

Early Growth Response gene 1 (Egr-1) regulates HSV-1 ICP4 and ICP22 gene expression

The molecular mechanisms mediating herpes simplex virus type 1 （HSV- 1） gene silencing during latent infection are not clear. Five copies of early growth response gene 1 （Egr-1） binding elements were identified in the intron of HSVol ICP22 （infected cell protein No. 22） gene, leading to the hypothesis that Egro 1 binds to the viral genome and regulates the viral gene expression. Transient co-transfection assays indicated that Egr-1 negatively regulated the transcription of both full-length and intron-removed ICP22 promoters. The same assays also revealed that Egr-1 repressed ICP4 （infected cell protein No. 4） promoter activity in a dose-dependent manner but showed less inhibition when the intron was removed. Histone deacetylation was not involved in this regulation since histone deacetylase inhibitor trichostatin A did not exhibit any effect on Egr-l-mediated repression. Chromatin immunoprecipitation assays showed that Egr-1 reduced the binding of Spl to the promoters and that the co-repressor Nab2 （NGFI-A/EGRl-binding protein） was recruited to the proximity of ICP4 in the presence of Egr-1. These results suggested that the multi functional transcription factor Egr-1 can repress HSV-1 immediate-early gene expression through the recruitment ofco-repressor Nab2 and reduction of Sp 1 occupancy, and thus may play a critical role in HSV-1 gene silencing during latency.

The Herpes Simplex Virus Type 1 Infected Cell Protein 22

As one of the immediate-early(IE)proteins of herpes simplex virus type 1(HSV-1),ICP22 is a multifunctional viral regulator that localizes in the nucleus of infected cells.It is required in experimental animal systems and some nonhuman cell lines,but not in Vero or HEp-2 cells.ICP22 is extensively phosphorylated by viral and cellular kinases and nucleotidylylated by casein kinase Ⅱ.It has been shown to be required for efficient expression of early(E)genes and a subset of late(L)genes.ICP22,in conjunction with the UL13 kinase,mediates the phosphorylation of RNA polymerase Ⅱ.Both ICP22 and UL13 are required for the activation of cdc2,the degradation of cyclins A and B and the acquisition of a new cdc2 partner,the UL42 DNA polymerase processivity factor.The cdc2-UL42 complex mediates postranscriptional modification of topoisomerase Ⅱα in an ICP22-dependent manner to promote L gene expression.In addition,ICP22 interacts with cdk9 in a Us3 kinase dependent fashion to phosphorylate RNA polymerase Ⅱ.

SPECIFIC IDENTIFICATION OF HERPES SIMPLEX VIRUS IN HUMAN ESOPHAGUS WITH RAPID IN SITU HYBRIDIZATION IN 5 CASES

HUMAN herpes simplex virus esophagitis （HSVE） was first reported in 1940 by Johnson. ^1HSVE usually occurs in immunocompromised patients,such as those with acquired immunodeficiency syndrome （AIDS）, 2-4 malignancies, cutaneous burns, connective tissue diseases, inflammatory bowel disease, those taking immuno-suppressive therapy, and those undergoing organ transplantation,5 etc. In the immunocompetent individuals, HSVE is rare, having been reported in 39 cases and mainly affecting young males^6,7 The aim of this study was to delineate the clinical experience in the diagnosis of HSVE using rapid in situ hybridization and assess the various detection methods.

Quantitation of Genital Herpes Virus DNA by Polymerase Chain Reaction and ELISA

Objective: To detect and quantitate genital herpes simplexvirus (HSV) DNA in specimens from 100 patients clinicallydiagnosed with genital herpes. Methods: Polymerase Chain Reaction (PCR) andenzyme-linked immunosorbent assay (ELISA) were used witha standard curve of DNA copies of HSV as quantitativecontrast. Results: Ninety-three cases were confirmed HSV positiveand 7 cases were found to be negative. There were 58 cases ofHSV-2 (62.4%) and 35 cases of HSV-1(37.6%) among the 93positive cases. The number of DNA plasmids ranged from 115to 1.1×10~5 per 250μL among the 93 positive samples (mean=7.1×10~4/250μL). The number of HSV DNA plasmids rangedfrom 136 to 1.1×10~5 copies per 250μL(mean=7.6×10~4) amongthose with HSV-2, and 115 to 9.4×10~4 per 250μL(mean=6.3×10~4) among those with HSV-1. Meanwhile 10μL ofextracted and dissolved DNA randomly taken from 8 each ofHSV-2 and HSV-1 samples were tested. The number of HSV-2DNA plasmids ranged from 35 copies to 2.7×10~4 (Mean=1.8×10~4) and the number of HSV-1 DNA ranged from 29 to2.5×10~4 (Mean=1.6×10~4). In the 7 negative cases, the quantityof HSV plasmids was zero. Conclusion: The sensitivity of ELISA quantitation (93%) isequal to that of Southern blot. The sensitivity of PCR fordiagnosis is 91%, and 88% for PCR typing.