| Human cytomegalovirus (HCMV), a member of the betaherpesvirinae subfamily, is a widespread pathogen responsible for generally asymptomatic and persistent infections in healthy people. However, it can cause severe disease illness and death in the absence of effective immune response among immunologically immature and immunocompromised individuals. Its negative impact, therefore, has increased in recent decades due to the rise in organ allografting, immunosuppressive treatment, and human immunodeficiency virus (HIV)-infected patients. Furthermore, it is the leading infectious agent causing birth defects.Human cytomegalovirus is one of the most complexes of animal viruses expressing a large number of proteins during the different virus replication period, which compromise the host immuno-defences. Little is known of the mechanisms underlying the pathogenicity of the virus. The detailed mechanisms involved have not yet been determined. Treatment of HCMV infections is difficult due to limited options. The current availabledrugs have produced significant clinical improvement, but suffer from poor oral bioavailability, low potency, drug resistance and dose-dependent toxicities. Anti-CMVdrugs include nucleoside analogues ganciclovir (GCV), cidofovir (CDV) and pyrophosphate analogue foscarnet (PFA) et al. With the improvement of basic medical research, many new antiviral medicines have been developed. Our institute has developed several anti-HCMV drugs which need to bescreened. Cytopathic Effect (CPE) assays are most frequently used approach to determinedrug sensitivities on virus with respect to antiviral clinical treatment ordrug design testing. Nevertheless, CPE is labor-intensive and time-consuming. Alternative tests have been established in the form of DNA-DNA hybridization methods and flow cytometry analyses, the latter providing the opportunity to analyze a large number of cells rapidly. Yet skill is necessary to achieve intracellular staining of viral antigens for flow cytometry, and protocols are restricted to specified applications. Therefore, effective and quick methods are required to screen the new antiviral elements.In this study, we have constructed recombinant green fluorescent protein-expressing human cytomegalovirus using molecular biology technologies as a tool to screen antiviral agents and to study the pathogenesis of HCMV. At first, we transformed the plasmid PHM673 to E coli. DH5a, and selected the positive clone, then amplified and purified the plasmid The plasmid pHM673, was linearized by using of restriction enzyme Nhel and transfected into HELF via LipofectamineTM 2000. After 24 hour of cultivation, cells were used for infection with 100TCID50 of HCMV strain Towne. Selection with Geneticin was started 24 hour postinfection. Following 3 weeks of passage in the presence of Geneticin, GFP fluorescence could be detected in most of the infected cells. Plaque assays were performed with infectious culture supernatant on HELF, and single virus plaques were grown by transfer to fresh HELF. DNA was isolated from infected HELF (fluorescence-positive wells) and confirmed for the presence of recombinant virus by PCR. The actions of different antiviral agents, different concentration of antiviral agents and different time were detected by recombinant virus.As a result, the recombinant green fluorescent protein-expressing human cytomegalovirus was successfully achieved. The GFP-based antiviral assay appeared to be very reliable for measuring virus-inhibitory effects in concentration- and time-dependent manners and might also be applied for high-throu.ghout screening of cytomegalovirus-specific antiviral agents. The antiviral elements developed by our medicine college and the Chinese traditional medicine are effective, which open up new possibilities for research application.
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