Expression Of NS1 Gene Research Articles

Association of Human Parvovirus B19 Infection with Development and Clinical Course of Myalgic Encephalomyelitis / Chronic Fatigue Syndrome

Abstract Our aim was to estimate the presence of B19V infection markers, the level of cytokines and time period since the appearance of infection in association with ME/CFS clinical symptoms. In 200 ME/CFS patients and 104 control group individuals the presence of B19V-specific IgG/IgM class antibodies, B19V NS1 gene sequence, mRNA expression, viral load and level of cytokines were determined. B19V-specific IgG-antibodies were found in 70% of ME/CFS patients and 67.4% of controls, IgM-antibodies in 8% of patients and in none of controls, B19V genomic sequences in 29% of patients and 3.8% of controls. 58.6% of positive patients had active and 41.4% had latent/persistent B19V infection. B19V NS1 gene expression was detected in 43% of patients. B19V load varied from < 0.2 copies to median 38.2 copies/µg of DNA. According to the antibody pattern, 36% of patients had a recent, and 43% had sustained B19V infection. Patients with the B19V genomic sequence and NS1 specific antibodies significantly more often had lymphadenopathy and multi-joint pain. Onset of the symptoms corresponded to time of appearance of B19V infection. IL-10 and TNF-levels were higher in patients with elevated B19V load. B19V genome 1 was identified in Latvian ME/CFS patients. The results indicated that at least in some cases B19V infection plays an important role in ME/CFS development

Influences of interaction between dengue virus type 2-infected human umbilical vein endothelial cells and macrophages on major inflammatory cytokines

Objective To study the influences on the production of major inflammatory cytokines after co-culturing macrophages with human umbilical vein endothelial cells (HUVECs) that were infected with dengue virus type 2 (DENV-2). Methods Density gradient centrifugation was used to isolate peripheral blood mononuclear cells (PBMC) from concentrated human leukocytes. Adherent monocytes in culture flasks were obtained and stimulated with macrophage colony-stimulating factor (M-CSF) to prepare macrophages. The purity of CD14+ CD11b+ cells was measured by flow cytometry. Changes in the expression of NS1 at mRNA level in HUVECs were detected by real-time PCR following DENV-2 infection. DENV-2-infected HUVECs were co-culture with macrophages in Transwell chambers. A control group was set up by pretreating HUVECs with sphingosine-1-phosphate (S1P) type 1 (S1P1)-specific receptor agonist CYM-5442 for 24 h to remove the drug before infection and then co-culturing the infected cells with macrophages. Real-time PCR was used to detect the expression at mRNA level of IL-6 and IL-8 in HUVECs and IL-6, IL-8, TNF-α and IL-1β in macrophages. A double-antibody sandwich ELISA was used to detect the expression of above cytokines in culture supernatants. Results After HUVECs were infected with DENV-2, expression of NS1 gene at mRNA level gradually increased to the peak at 24 h (2.66±0.53, P<0.05) and then decreased. The purity of macrophages detected by flow cytometry was (89.16±2.07) %. Expression of IL-6 and IL-8 at mRNA level in DENV-2-infected HUVECs was up-regulated. The peak values reached at 24 h of IL-6 and IL-8 expression were 16.10±0.17 and 29.76±0.58, while the expression levels at 24 h in the uninfected group were 1.46±0.67 and 1.60±0.54, respectively. Expression of IL-6, IL-8, TNF-α and IL-1β at mRNA level in DENV-2-infected macrophages was increased significantly. The levels of IL-6, IL-8, TNF-α and IL-1β expression at 24 h were 45.82±3.72, 52.34±1.69 (12 h), 8.94±1.75 and 30.96±1.44 in the infected macrophages, and 1.16±0.22, 1.15±0.21, 1.11±0.09 and 1.47±0.31 in the uninfected group. Expression of these cytokines was decreased at every time points after co-culturing of DENV-2-infected HUVECs with macrophages, but still significantly higher than that in the uninfected group. In the co-culture group with DENV-2 infection, CYM-5442 pretreatment significantly decreased the expression at mRNA level of IL-6 and IL-8 in HUVECs (P<0.01) and that of IL-6, IL-8, TNF-α and IL-1β in macrophages (P<0.01). Conclusions DENV-2 could infect primary HUVECs, and then activate macrophages to promote the secretion of large amounts of IL-6, IL-8, TNF-α and IL-1β. Moreover, the activated macrophages could reduce the production of inflammatory cytokines in HUVECs to a certain extent. Key words: Dengue virus type 2 (DENV-2); Human umbilical vein endothelial cell (HUVEC); Macrophage; Cytokine; Immunoregulation

Validation of Interferon Induced IFIT Genes in Host Antiviral Defence Against BTV16 Infection : An Initial Report

Whole blood samples were collected from healthy sheep and goat, Peripheral blood mononuclear cells (PBMC) isolation was done from whole blood of both the species and cultured in six well plates using RPMI-1640 media in the presence of 5% CO2 at 37 0C. Bluetongue (virus BTV)-16 infection was given in PBMCs of both the species at 0 h and representative samples were collected at 24, 48 and 72 h. Total RNA was extracted and cDNA was obtained by reverse transcription. All the four differentially expressed genes (IFITs genes) were validated using quantitative real time PCR and the target gene expression was studied in fold change at 24, 48 and 72 h post infection of sheep and goat PBMCs against BTV-16 infection along with mock infected cells as control. Up regulation in target gene expression was found in virus infected PBMCs of both the species at all the time intervals than control PBMCs indicates the antiviral host protective response of those DEGs against BTV-16 infection. NS1 gene expression of BTV16 in sheep and goat PBMCs at different time interval were also determined which showed significant variation in both species.

Comprehensive traditional Chinese medicine intervention for perimenopausal syndrome in women: a community study

As perimenopausal syndrome is a particularly disturbing condition to the patient, it is practical and necessary to establish a program of comprehensive traditional Chinese medicine therapy for women with perimenopausal syndrome. To observe the therapeutic effects of a comprehensive traditional Chinese medicine therapy for women with perimenopausal syndrome in the community. Women with perimenopausal syndrome who met the inclusion criteria in 3 communities of Shanghai were selected for this study. Comprehensive traditional Chinese medicine therapy, including administration of Chinese herbs, auricular point therapy, psychological counseling and the practice of Tai Chi, was applied for these women. The modified Kupperman index was measured once every 4 weeks for 12 weeks; the indexes of menopause-specific quality of life (MENQOL) questionnaire were measured before and after the treatment. There was no significant difference in 4 groups of negative conversion ratio of the modified Kupperman index at the 4th, 8th and 12th week, respectively. The indexes of the MENQOL questionnaire showed significant differences in 4 groups before and after the treatment. The overall efficacy rate of the comprehensive traditional Chinese medicine therapy in women with perimenopausal syndrome achieved 97.84% at the 8th and the 12th week. The comprehensive traditional Chinese medicine therapy can achieve significant improvements in women with perimenopausal syndrome who exhibit both physical and psychological symptoms. With such significant clinical effects, the comprehensive traditional Chinese medicine therapy should be promoted in the community.

Investigation of the sensitivities of distinct gastric cancer cells to parvovirus H‐1 induced cytotoxicity

To investigate the sensitivities of distinct gastric cancer cells to parvovirus H-1 induced cytotoxicity and the possible mechanism(s). There were six distinct differentiated gastric cancer cell lines: HGC27 (undifferentiated), BGC823 (undifferentiated), MKN45 (poorly differentiated), AGS (poorly differentiated), SGC7901 (moderately differentiated) and MKN28 (well differentiated). The cell cycle distributions were measured by flow cytometry and the differential sensitivities of the six distinct gastric cancer cells after H-1 virus infection were detected by MTT assay. RT-PCR was used to detect viral NS1 gene expression in all six gastric cancer cell lines. The S phase ratios of HGC27, BGC823, MKN45, AGS, SGC7901 and MKN28 were 24.72%, 30.15%, 27.10%, 29.03%, 31.82% and 33.73%, respectively. HGC27 cells were sensitive to H-1 virus induced cytotoxicity, followed by SGC7901 cells. MKN45 and AGS cells were moderately sensitive and MKN28 cells were insensitive. However, BGC823 cells were resistant to H-1 virus induced cytotoxicity. The expressions of viral NS1 were higher in HGC27, BGC823, MKN45 and SGC7901 cells, and lower in AGS and MKN28 cells. The sensitivities of the distinct gastric cancer cells to H-1 virus induced cytotoxicity were markedly different. In general, the poorly differentiated cells showed an enhanced sensitivity to H-1 virus attack compared with well-differentiated ones. The enhanced sensitivity of poorly versus well-differentiated gastric cancer cells to H-1 virus is related in part to the enhanced capacity of the former for NS1 protein production and accumulation. The undifferentiated BGC823 cells were resistant to H-1 virus triggered cytotoxicity. It may further verify that not all tumor cells are sensitive to H-1 virus lytic effects.

Tick-borne encephalitis virus NS1 glycoprotein during acute and persistent infection of cells

Tick-borne encephalitis virus (TBEV) was propagated in porcine embryo kidney (PS) cells until 48 h whereas human kidney (RH) cells maintained the virus persistence during at least 2 months. One of possible reasons of flavivirus chronic infection might be abnormal NS1 gene expression. Immunoblotting with monoclonal antibodies (MAbs) revealed the similarity of the intracellular and secreted NS1 nonstructural glycoprotein size and linear antigenic determinants in both the infected cell lines. However, according to the competitive binding of MAbs with the TBEV NS1 extracellular glycoprotein, its contiguous epitopes differed for acute or persistent infection. To map the TBEV NS1 glycoprotein antigenic determinants its recombinant analogues were used. All the studied MAbs could bind with the full-length NS1 recombinant protein. Deletion of the TBEV NS1 gene internal region resulted in defective NS1d1 protein without the region between 269 and 333 a.a. Lack of NS1d1 binding with 20B4 MAb and diminished binding with 22H8 and 17C3 MAbs permitted to map their antigenic determinants within or nearby deleted region, respectively. Interaction of other MAbs with the NS1 and NS1d1 recombinant proteins did not differ, suggesting that their epitopes were located in the region of N-terminal 268 a.a. or C-terminal 19 a.a. of the TBEV NS1 protein. The second NS1d2 truncated protein contained the first N-terminal 33 a.a. of the TBEV NS1 protein and was able to bind with 29G9 MAb. Taken together the data stand for the differences in the N-terminal structure of the TBEV NS1 multimers secreted from the acute and persistent infected cells whereas the intracellular and secreted monomer processing was the same. The modified NS1 protein oligomers in the RH cellular line might slow virus replication and could result in the TBEV persistence.

Expression of the Tick-borne Encephalitis Virus NS1 Gene in Gram-Negative Bacteria from the Mouse Nasopharynx

Bacteria were isolated from the nasopharynx of BALB/c mice and electroporated with pUR290(NS1)2 containing two copies of tick-borne encephalitis virus (TBEV, strain Sofjin) NS1 under the control of the lac promoter. The plasmid persisted in transformants for at least ten passages. The NS1 gene expression was detected in Gram-negative enterobacteria by immunoblotting with monoclonal antibodies against the TBEV nonstructural glycoprotein NS1. Recombinant NS1 was detected in bacterial cells and in the culture medium. Intranasal immunization with recombinant bacteria caused antibodies against NS1 to appear in the serum of BALB/c mice. However, the humoral immune response to NS1 failed to protect mice from a TBEV challenge.

A preliminary analysis of antineoplastic activity of parvovirus MVMp NS-1 proteins.

Human gastric cancer MKN-45 cells were transfected with pULB 3238, a plasmid carrying MVMp NS-1 gene with its original P4 promoter replaced by the glucocorticoid inducible promoter MMTV-LTR. After the integration and expression of NS-1 gene, some of the transfectants died, while others remained alive, but the growth features of survived cells were changed. For further study on the antineoplastic function of parvoviral NS-1 protein in vivo, transgenic mice carrying NS-1 genes were established by conventional method. Among 4 founders, one of them was found to be able to transmit the transgene to around 50% of their offsprings. RT-PCR was performed to indicate the expression of NS-1 gene in transgenic mice and its mRNA appeared in a variety of tissues. The expression of integrated NS-1 gene may correlate with the decreased incidence of tumor induced in vivo by chemical carcinogens.

A primer vector system that allows temperature dependent gene amplification and expression in mammalian cells: regulation of the influenza virus NS1 gene expression.

Influenza virus RNA segment 8 has been cloned into primer-vector pSLts1. This vector was designed to replicate in simian cells in a temperature dependent fashion by use of the SV40 tsA209 T-antigen gene. The oriented synthesis of cDNA on dT-tailed pSLts1 was performed on in vitro synthesized mRNA, and the second DNA strand was primed with an influenza-specific terminal oligodeoxynucleotide. Recombinant pSLVa232 contained the RNA segment 8 sequence directly fused to the SV40 late promoter contained in pSLts1, and followed by the SV40 polyadenylation signal. Expression of NS1 gene in transfected COS cells took place at a level comparable to that found in infected cells. When VERO cell cultures were transfected with recombinant pSLVa232, expression of the NS1 gene was temperature dependent. Close to one hundred fold increase in the amplification and expression of the cloned gene was observed after shift down of the transfected cells to permissive temperature. Vector pSLts1 and the cloning strategy described may be useful for the specific cloning and regulated expression of mRNAs of known 5'-terminal sequence.