[Antiviral activity of nano-realgar against herpes simplex virus Type II in vitro].

OBJECTIVE: To explore the antiviral activity of nano-realgar against herpes simplex virus Type II (HSV-2) in vitro.
 Methods: Acyclovir (ACV) as a positive control, the cytotoxicity of nano-realgar at different concentrations (including 200.00, 150.00, 100.00, 50.00, 25.00, 12.50, 6.25, 3.13, 1.54, 0.78, 0.39 and 0 mg/L) on normal Vero cells were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. HSV-2 virus titer was determined by plaque assay, and the Vero cells model of HSV-2 infection was established. Subsequently, the antiviral effects of nano-realgar at different concentrations (including 20.00, 10.00, 5.00, 2.50, 1.25, 0.63, 0.31, 0.15, 0.08, 0.04 and 0 mg/L) on infected cells model were evaluated by the observation of cytopathic effect (CPE) and MTT method under the 3 modes including pre-treatment, treatment and direct inactivation.
 Results: The 50% cytotoxic concentration (CC50) of nano-realgar on Vero cells was 37.15 mg/L. The titer of HSV-2 was 7.30 log PFUs/mL. In the 3 modes, the half-maximal effective concentration (EC50) of nano-realgar on HSV-2 infected Vero cells were 0.13, 1.80 and 0.52 mg/L, and the corresponding therapeutic index (TI) were 285.77, 20.64, 71.44, respectively. The TI value of nano-realgar on pre-treatment mode was higher than that of nano-realgar on treatment and direct inactivation modes.
 Conclusion: Nano-realgar can play a good anti-HSV-2 activity in the 3 modes (pre-treatment, treatment and direct inactivation), and the anti-HSV-2 efficacy of nano-realgar on pre-treatment mode is better than that of nano-realagr on other 2 modes.

The Long Noncoding RNA NEAT1 Exerts Antihantaviral Effects by Acting as Positive Feedback for RIG-I Signaling.

Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs (lncRNAs) play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus (HTNV) infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon (IFN-ß) production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I-IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein (SFPQ) by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs (lncRNAs) as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon (IFN) responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I-IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.

Soluble expression and purification of Brucella cell surface protein (BCSP31) of Brucella melitensis and preparation of anti-BCSP31 monoclonal antibodies.

Brucella cell surface protein (BCSP31) is potentially useful for diagnosing brucellosis. We aimed to establish a monoclonal antibody (MAb) against Brucella melitensis BCSP31 and to investigate its distribution in diagnosis. Soluble recombinant BCSP31 was successfully expressed and purified. Two MAbs (1F1 and 1E5) against B. melitensis BCSP31, effective in detecting both recombinant and cellular proteins, were obtained and characterized. The MAbs did not react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus aeruginosus, but strongly reacted with BCSP31 and B. melitensis by ELISA and Western blot analysis. We also tested different Brucella species and brucellosis using the prepared anti-BCSP31 MAbs. BCSP31 and anti-BCSP31 MAbs may play important roles in future research in diagnosing brucellosis.

Immune Efficacy of a Genetically Engineered Vaccine against Lymphocystis Disease Virus: Analysis of Different Immunization Strategies.

Here, we report the construction of a vaccine against lymphocystis disease virus (LCDV) using nucleic acid vaccination technology. A fragment of the major capsid protein encoding gene from an LCDV isolated from China (LCDV-cn) was cloned into an eukaryotic expression vector pEGFP-N2, yielding a recombinant plasmid pEGFP-N2-LCDV-cn0.6 kb. This plasmid was immediately expressed after liposomal transfer into the Japanese flounder embryo cell line. The recombinant plasmid was inoculated into Japanese flounder via two routes (intramuscular injection and hypodermic injection) at three doses (0.1, 5, and 15 µg), and then T-lymphopoiesis in different tissues and antibodies raised against LCDV were evaluated. The results indicated that this recombinant plasmid induced unique humoral or cell-mediated immune responses depending on the inoculation route and conferred immune protection. Furthermore, the humoral immune responses and protective effects were significantly increased at higher vaccine doses via the two injection routes. Plasmid pEGFP-N2-LCDV0.6 kb is therefore a promising vaccine candidate against LCDV in Japanese flounder.

Viruses Run: The Evasion Mechanisms of the Antiviral Innate Immunity by Hantavirus.

Hantavirus can cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome (HPS) in America, with high mortality and unknown mechanisms. Innate immunity is the host's first-line defense to bridge the acquired immunity against viral infections. However, hantavirus has evolved various strategies in both molecular and cellular aspects to evade the host's natural immune surveillance. The Interferon-I (IFN-I) signaling pathway, a central link of host defense, induces various antiviral proteins to control the infection. This paper summarizes the molecular mechanisms of hantavirus evasion mechanisms of the IFN signaling pathway and cellular processes such as regulated cell death and cell stress. Besides, hantavirus could also evade immune surveillance evasion through cellular mechanisms, such as upregulating immune checkpoint molecules interfering with viral infections. Understanding hantavirus's antiviral immune evasion mechanisms will deepen our understanding of its pathogenesis and help us develop more effective methods to control and eliminate hantavirus.

[Research progress in metabolic reprogramming of endothelial cells and T cells and potential therapeutic targets for chronic inflammation].

Metabolic reprogramming plays a very important role in the immunoregulatory process, and T cells, as the indispensable part in the immune response, realize the change of function and state through metabolic reprogramming. And endothelial cells exhibit similar metabolic reprogramming. This review explores the interaction between endothelial cells and T cells to reveal the mechanism of the former as non-professional antigen presenting cells to recruit and activate the latter and the specific mechanism of cytokines produced by the latter in inflammatory response to regulate the function and state of the former, aiming to find the potential therapeutic targets for chronic inflammation and provide new ideas for the treatment.

The value of serum visfatin in predicting in-stent restenosis of drug-eluting stents.

OBJECTIVE: This study aims to determine the association between serum visfatin level and in-stent restenosis (ISR) after percutaneous coronary intervention (PCI) using drug-eluting stents (DES). METHODS: A total of 460 patients with stable coronary heart disease who underwent DES placement were included. According to the results of coronary angiography 1year after PCI, 62 patients diagnosed as ISR were enrolled into the ISR group and 398 patients without ISR were recruited into the control group. Baseline clinical data were collected, and serum visfatin level was measured using ELISA method. RESULTS: The serum visfatin levels before PCI were not different between ISR and control groups (P=0.41). However, visfatin level after PCI in the ISR group was significantly higher than in the control group [(30.2±8.6) ng/ml vs. (22.6±7.9) ng/ml, P<0.01]. In multivariate logistic regression, the independent predictors for ISR included post-procedural visfatin level (odds ratio [OR]: 2.08, 95% confidence interval [CI]: 1.19-3.65), type 2 diabetes (OR: 2.30, 95% CI: 1.10-4.79), reference vessel diameter (OR: 0.79, 95% CI: 0.63-0.98), stent length (OR: 1.52, 95% CI: 1.05-2.21) and stent diameter (OR: 0.67, 95% CI: 0.51-0.88). ROC curve analysis indicated that the area under the curve for post-procedural visfatin in predicting ISR was 0.82 (95% CI: 0.77-0.86), with the optimal cut-off value of 25.9ng/ml showing a sensitivity of 84.0% and a specificity of 69.3%. CONCLUSION: Increased serum visfatin level after DES placement is independently associated with ISR. Serum visfatin may be useful in the prediction of ISR.

In-Cell Western Assays to Evaluate Hantaan Virus Replication as a Novel Approach to Screen Antiviral Molecules and Detect Neutralizing Antibody Titers.

Hantaviruses encompass rodent-borne zoonotic pathogens that cause severe hemorrhagic fever disease with high mortality rates in humans. Detection of infectious virus titer lays a solid foundation for virology and immunology researches. Canonical methods to assess viral titers rely on visible cytopathic effects (CPE), but Hantaan virus (HTNV, the prototype hantavirus) maintains a relatively sluggish life cycle and does not produce CPE in cell culture. Here, an in-cell Western (ICW) assay was utilized to rapidly measure the expression of viral proteins in infected cells and to establish a novel approach to detect viral titers. Compared with classical approaches, the ICW assay is accurate and time- and cost-effective. Furthermore, the ICW assay provided a high-throughput platform to screen and identify antiviral molecules. Potential antiviral roles of several DExD/H box helicase family members were investigated using the ICW assay, and the results indicated that DDX21 and DDX60 reinforced IFN responses and exerted anti-hantaviral effects, whereas DDX50 probably promoted HTNV replication. Additionally, the ICW assay was also applied to assess NAb titers in patients and vaccine recipients. Patients with prompt production of NAbs tended to have favorable disease outcomes. Modest NAb titers were found in vaccinees, indicating that current vaccines still require improvements as they cannot prime host humoral immunity with high efficiency. Taken together, our results indicate that the use of the ICW assay to evaluate non-CPE Hantaan virus titer demonstrates a significant improvement over current infectivity approaches and a novel technique to screen antiviral molecules and detect NAb efficacies.

Incorporation of GM-CSF or CD40L Enhances the Immunogenicity of Hantaan Virus-Like Particles.

A safe and effective Hantaan virus (HTNV) vaccine is highly desirable because HTNV causes an acute and often fatal disease (hemorrhagic fever with renal syndrome, HFRS). Since the immunity of the inactivated vaccine is weak and the safety is poor, HTNV virus-like particles (VLPs) offer an attractive and safe alternative. These particles lack the viral genome but are perceived by the immune system as virus particles. We hypothesized that adding immunostimulatory signals to VLPs would enhance their efficacy. To accomplish this enhancement, we generated chimeric HTNV VLPs containing glycosylphosphatidylinositol (GPI)-anchored granulocyte macrophage colony-stimulating factor (GM-CSF) or CD40 ligand (CD40L) and investigated their biological activity in vitro. The immunization of mice with chimeric HTNV VLPs containing GM-CSF or CD40L induced stronger humoral immune responses and cellular immune responses compared to the HTNV VLPs and Chinese commercial inactivated hantavirus vaccine. Chimeric HTNV VLPs containing GM-CSF or CD40L also protected mice from an HTNV challenge. Altogether, our results suggest that anchoring immunostimulatory molecules into HTNV VLPs can be a potential approach for the control and prevention of HFRS.

Development of a SYBR-Green â quantitative PCR assay for the detection and genotyping of different hantaviruses.

Hemorrhagic fever with renal syndrome (HFRS) is a severe, viral zoonotic disease which occurs worldwide, particularly in Asia and Europe. In China, the Hantaan virus (HTNV) and the Seoul virus (SEOV) are known to be the most prevalent causative agents of HFRS. Since no protective vaccines or effective treatments are available for human use, accurate and reliable diagnostic methods are essential for disease surveillance. In the present study, the viral loads in cell culture supernatant, infected mice blood and clinical serum samples were quantified using the SYBR­Green I-based reverse transcription-quantitiative polymerase chain reaction (RT-qPCR) assay, which targeted the S gene sequence of the HTNV and SEOV genomes. The cRNA of these two viruses were synthesized as a positive control and 10-fold serially diluted from 1x105 to 1x100 copies/µl. Standard curves were generated by plotting the mean cycle threshold (Ct) values versus copy numbers. The standard curve of HTNV had a correlation coefficient (R2) of 0.994, efficiency of amplification (E) of 101.9%, and the slope of -3.278, whereas that of SEOV had an R2 of 0.993, E of 104.8%, and the slope of -3.212. The minimum detection limit of the RT-qPCR assay for HTNV and SEOV was 101 copies/µl. Two qPCR assays were successfully established for the detection of HTNV and SEOV, respectively. Taken together, these findings demonstrate that using the SYBR­Green I-based RT-qPCR assay, the HTNV and SEOV may be genotyped precisely without cross-reactivity. Furthermore, viral RNA may be detected and quantified in cells, mice and infected individuals, which may be useful in epidemiological studies as well as for early monitoring and further preventative treatment against SEOV and HTNV-induced diseases.

Screening and Identification of an H-2Kb-Restricted CTL Epitope within the Glycoprotein of Hantaan Virus.

The cytotoxic T lymphocyte (CTL) response plays a key role in controlling viral infection, but only a few epitopes within the HTNV glycoprotein (GP) that are recognized by CTLs have been reported. In this study, we identified one murine HTNV GP-derived H2-Kb-restricted CTL epitope in C57BL/6 mice, which could be used to design preclinical studies of vaccines for HTNV infection. First, 15 8-mer peptides were selected from the HTNV GP amino acid sequence based on a percentile rank of <=1% by IEDB which is the most comprehensive collection of epitope prediction and analysis tool. A lower percentile rank indicates higher affinity and higher immune response. In the case of the consensus method, we also evaluated the binding score of peptide-binding affinity by the BIMAS software to confirm that all peptides were able to bind H2-Kb. Second, one novel GP-derived CTL epitope, GP6 aa456-aa463 (ITSLFSLL), was identified in the splenocytes of HTNV-infected mice using the IFN-γ ELISPOT assay. Third, a single peptide vaccine was administered to C57BL/6 mice to evaluate the immunogenic potential of the identified peptides. ELISPOT and cell-mediated cytotoxicity assays showed that this peptide vaccine induced a strong IFN-γ response and potent cytotoxicity in immunized mice. Last, we demonstrated that the peptide-vaccinated mice had partial protection from challenge with HTNV. In conclusion, we identified an H2-Kb-restricted CTL epitope with involvement in the host immune response to HTNV infection.

Expression of angiostatin cDNA in human hepatocellular carcinoma cell line SMMC-7721 and its effect on implanted carcinoma in nude mice.

AIM: To transfect murine angiostatin cDNA into human hepatocellular carcinoma cell line SMMC-7721 and to investigate its effects on implanted carcinoma in nude mice. METHODS: A eukaryotic expression vector of pcDNA3.1-mAST containing murine angiostatin was constructed. Then pcDNA3.1-mAST plasmid was transfected into cell line SMMC-7721 by Lipofectamine. The resistant clone was screened by G418 filtration and identified by RT-PCR and Western blotting. Nude mice were divided into three groups of 10 each. Mice in blank control group were only injected with SMMC-7721 cells. Mice in vector control group were injected with SMMC-7721 cells transfected with pcDNA3.1 (+) vector, whereas mice in angiostatin group were injected with SMMC-7721 cells transfected with pcDNA3.1-mAST plasmid. Volume, mass and microvessel density (MVD) of the tumors in different groups were measured and compared. RESULTS: Murine angiostatin cDNA was successfully cloned into the eukaryotic expression vector pcDNA3.1 (+). pcDNA3.1-mAST was successfully transfected into SMMC-7721 cell line and showed stable expression in this cell line. No significant difference was observed in the growth speed of SMMC-7721 cells between groups transfected with and without angiostatin cDNA. Tumor volume, mass and MVD in the angiostatin group were significantly lower than those in the blank control group and vector control group (P<0.01). The inhibitory rate of tumor reached 78.6%. Mass and MVD of the tumors only accounted for 34.6% and 48.9% respectively of those in the blank control group. CONCLUSION: Angiostatin cDNA could be stably expressed in human hepatocellular carcinoma cell line SMMC-7721 without obvious inhibitory effects on the growth of SMMC-7721 cells. When implanted into nude mice, SMMC-7721 cells transfected with angiostatin cDNA show a decreased tumorigenic capability. It suggests that angiostatin can inhibit tumor growth through its inhibition on angiogenesis in tumors.

[Inhibitory effects of murine angiostatin on implant carcinoma of nude mouse].

OBJECTIVE: To study the effects of murine angiostatin, which was transfected into the human hepatocellular cancer cell line SMMC-7721, on the implant carcinoma of nude mouse. METHODS: The human hepatocellular cancer cell line SMMC-7721, which could express murine angiostatin gene stably, was constructed. The animals were divided into three groups: SMMC-7721 cell was implanted into control group, SMMC-7721/pcDNA3.1 (+) cell was implanted into vector group, and SMMC-7721/pcDNA3.1-mAST cell was implanted into angiostatin group. The carcinoma volume, weight, and microvessel density (MVD) of each group were compared. RESULTS: The implant carcinoma volume in 35 days was (3 538.1 +/- 643.3) mm(3), (3 128.5 +/- 546.6) mm(3), and (755.8 +/- 198.2) mm(3) in the control group, vector group, and angiostatin group. The carcinoma weight of the control group, vector group, and angiostatin group was (6.0 +/- 0.7) g, (5.9 +/- 0.5) g, (2.1 +/- 0.5) g, respectively. The carcinoma MVD was 52.2 +/- 6.6, 49.4 +/- 7.0, and 25.5 +/- 4.1 accordingly. The carcinoma volume, weight, and MVD of the angiostatin group were significantly smaller than those of the control group and vector group (P < 0.01). The inhibitory rate of carcinoma reached 78.6%. CONCLUSIONS: Nude mouse experiments showed that the tumorigenic capacity of cells transfected had been reduced greatly, and that the carcinoma volume, weight and MVD were significantly lower than those of the control group. We conclude that angiostatin inhibits the growth of carcinoma by its inhibition of carcinoma angiogenesis.

Recombinant fusion proteins FPTD-Grb2-SH2 and FPTD-Grb2-SH2M inhibit the proliferation of breast cancer cells in vitro.

Growth factor receptor bound protein 2 (Grb2) is a key adaptor performing a principal role in the oncogenic Ras signaling pathway. In the present study, we generated two fusion proteins. One contained an Src homology 2 (SH2) domain of Grb2, a signal peptide sequence, FLAG-tag sequence, PTD region and we named it FPTD-Grb2-SH2, while the other contained one mutant SH2 domain, added to a signal peptide sequence, FLAG-tag sequence, PTD region and we named it FPTD-Grb2-SH2M. Western blot analysis and immunofluorescence assay were used to investigate the expression and location of the fusion proteins in breast cancer cells. The proliferation and migration of the cells were estimated by MTT and Transwell cell migration assays, respectively. Flow cytometric analysis was performed to evaluate the apoptosis of the breast cancer cells. The recombinant proteins FPTD-Grb2-SH2 and FPTD-Grb2-SH2M were successfully expressed in the breast cancer cell lines regardless of HER2-phenotype, and they suppressed breast cancer cell growth and migration as expected from the lack of SH3 domain. Both FPTD-Grb2-SH2 and FPTD-Grb2-SH2M exhibited significant toxicity to breast cancer cells. The present study demonstrated that the recombinant proteins FPTD-Grb2-SH2 and FPTD-Grb2-SH2M may be used for anticancer drug development.

AhR expression is increased in hepatocellular carcinoma.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor implicated in multiple cellular processes and its expression has been shown to play a critical role in tumorigenesis. However, the role of AhR in tumorigenesis of hepatocellular carcinoma remains unclear. In the current study, we investigated the role of AhR in hepatocellular carcinoma tumorigenesis and progression by (a) measuring the expression levels of AhR in liver lesions and (b) assessing the correlation between AhR expression and clinicopathologic parameters. The tissue microarray used in this study contained hepatocellular carcinoma tissues (n = 94), cancer adjacent normal hepatic tissues (n = 5) and normal hepatic tissues (n = 5), which were immunohistochemically assessed for AhR expression. Significantly stronger AhR staining was observed for hepatocellular carcinoma tissues than for cancer adjacent normal hepatic tissues (P = 0.003) and normal hepatic tissues (P = 0.004). In addition, AhR expression was associated with T stage (P = 0.03). The results from this study suggest that an increase in AhR expression is associated with hepatocellular carcinoma progression and may have a potential role in the treatment of hepatocellular carcinoma.

Induction of Hantaan virus-specific immune responses in C57BL/6 mice by immunization with a modified recombinant adenovirus containing the chimeric gene, GcS0.7.

Hantavirus glycoprotein Gc is one of the main components that contribute to the generation of humoral immune responses, while the nucleocapsid protein (NP) is involved in cellular immune responses through the induction of antibody-dependent cytotoxic T cells. In this study, a chimeric gene, GcS0.7, which encodes a fusion protein containing Gc and truncated NP, was constructed as a candidate for Hantaan virus (HTNV) vaccine development. The chimeric gene was cloned into an adenoviral vector in conjunction with the powerful hybrid cytomegalovirus (CMV) enhancer/chicken ß-actin (CAG) promoter or the woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (WPRE), or both. Both elements increased the expression level of the fusion protein. The rAd-GcS0.7-pCAG group demonstrated the highest fusion protein expression level, with a 2.3-fold increase compared with the unmodified adenoviral vector. To further evaluate the humoral and cellular immunity induced by the recombinant adenovirus, the antibody titers, interferon (IFN)-γ secretion level and cytotoxic T cell ratio were detected in immunized mice. The strongest HTNV­specific humoral and cellular immune responses were detected in the rAd-GcS0.7­pCAG group. The immunogenicity of these recombinant adenoviruses was compared with that of the inactivated vaccine through a series of immunological assays. In terms of the cellular immune responses, the rAd-GcS0.7-pCAG group even exceeded those induced by the vaccine control. The CAG hybrid promoter improved not only the expression level, but also the immunogenicity of the fusion protein, and may thus provide a promising strategy for HTNV vaccine research.

Modification of the adenoviral transfer vector enhances expression of the Hantavirus fusion protein GnS0.7 and induces a strong immune response in C57BL/6 mice.

Hantavirus glycoproteins (Gn and Gc) are significant components of vaccines for haemorrhagic fever with renal syndrome (HFRS); however, they are not effective due to weak immunogenicity and low levels of production in expression systems. To circumvent this problem, a 0.7-kb fragment of the S segment was fused to Gn, and a hybrid CAG promoter/enhancer in conjunction with (or without) the WPRE (Woodchuck hepatitis virus post-transcriptional regulatory element) was used to improve the expression of fusion protein GnS0.7 in the adenoviral expression system. The expression level of the fusion protein as well as the response of mice immunized with recombinant adenoviruses containing GnS0.7 was investigated. In addition, a series of immunological assays were conducted to determine the immunogenicity of the recombinant adenoviruses. The results showed that the recombinant adenovirus with the CAG promoter/enhancer (rAd-GnS0.7-pCAG) expressed approximately 2.1-fold more GnS0.7 than the unmodified recombinant adenovirus containing GnS0.7 (rAd-GnS0.7-pShuttle). This enhanced expression level was also higher than for other modified recombinant adenoviruses studied. Animal experiments showed that rAd-GnS0.7-pCAG induced a stronger Hantaan virus (HTNV)-specific humoral and cellular immune response in mice, with the cellular immune response to the GnS0.7 being stronger than the HFRS vaccine control. These results demonstrate that the CAG promoter/enhancer improved significantly the expression of the chimeric gene GnS0.7 in the adenovirus expression system. These findings may have significant implications for the development of genetically engineered vaccines for HFRS.

[Construction and expression of the eukaryotic expression vector carrying HSV-1 gC glycoprotein gene].

AIM: To stably express herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) in Chinese hamster ovary cells (CHO-K1). METHODS: The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed and transfected into CHO-K1 cells by Lipofectamine 2000. The transfected cells were selected by G418 and methotrexate (MTX). The expression of HSV-1 gC was analyzed by Slot blot. HSV-1 gC proteins were purified with His-Ni Sepharose and then detected by Western blot. RESULTS: The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed successfully. CHO-K1 cells stably expressing HSV-1 gC proteins were established and confirmed by Western blot. CONCLUSION: The HSV-1 gC proteins have been expressed successfully and have good bioactivity. The results make it possible for further study and clinical use of HSV-1 gC.

[Construction and immunogenic study of recombinant adenovirus containing chimeric gene G2S0.7 and CTL epitopes of Hantaan virus].

AIM: To express G2 fragment of M segment and 0.7 kb fragment of S segment and several CTL epitopes of S segment in adenovirus expression system and investigate the immunological properties of hantaan virus chimeric gene. METHODS: The recombinant adenovirus was constructed and the recombinant adenovirus was obtained after transfecting HEK293 cells. The titer of it was determined and the expressed product was detected by IFA and ELISA. Further, BALB/c mice were vaccinated by the recombinant adenovirus and the immune response was tested by ELISA, microcell-culture neutralizing experiment, T lymphocyte proliferation test (MTT assay) and cell-mediated cytotoxicity assay. RESULTS: The recombinant adenovirus AG2S0.7CTL1, AG2S0.7CTL2 were constructed successfully and the titer of it was about 10¹°-10¹¹ pfu/mL. The expressed protein could be recognized by the hantaan virus NP-specific mAb and glycoprotein G2-specific mAb. The recombinant adenovirus containing CTL epitopes could elicit effectively the cellular immune response aimed to the NP and GP of hantaan virus in BALB/c mice. CONCLUSION: The recombinant adenovirus containing CTL epitopes could induce the higher cellular immune response than the group that not containing CTL epitopes.

[Construction and identification of a new type of recombinant adenovirus containing S0.7 gene of Hantavirus].

AIM: To construct a adenovirus vector containing the 0.7 kb fragment of S gene of Hantavirus, CAG promoter, and WPRE (mRNA-stabilizing post-transcriptional regulatory element from the woodchuck hepatitis virus). METHODS: The fragments of CAG and WPRE were synthesized according to GenBank, and inserted into the plasmid pShuttle-S0.7 to create a transfer vector pShuttle-S0.7-CAG-WPRE. The S0.7-CAG-WPRE fragment was then cloned into Adeno-X; Viral DNA by PI-Sce I and I-Ceu I digestion. The recombinant adenovirus DNA was linearized by Pac I, transfected into HEK 293 cells via Lipofectamine; 2000, and the titer of the recombinant adenovirus was determined by Adeno-X; Rapid Titer Kit. The expressed product of S0.7-CAG-WPRE fragment was detected by immunofluorescence assay. RESULTS: The sequence of S0.7-CAG-WPRE fragment was confirmed by sequencing, and the recombinant adenovirus containing S0.7-CAG-WPRE was titered at 10(13); pfu/L. HEK293 cells transfected with recombinant adenoviruses were detected positive by immunofluorescence assay using a specific mAb 1A8 against Hantavirus nucleoprotein. CONCLUSION: The recombinant adenovirus containing the 0.7 kb fragment of S segment of Hantavirus, CAG promoter, WPRE was constructed.