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Dengue virus (dengue virus, DV) is a single strand of the genus Flavivirus positive-strand RNA virus, four serotypes (DV1 ~ 4). DV primarily by Aedes aegypti and Aedes albopictus spread, each serotype can cause human dengue (classical dengue fever, DF) and dengue hemorrhagic fever / dengue shock syndrome (dengue hemorrhagic fever / dengue shock syndrome, DHF / DSS). In recent years, with the development of tourism and global warming, DF and DHF / DSS epidemic outbreaks more frequent. DHF / DSS patients with high mortality, the pathogenesis is unclear. Multiple clinical studies have shown that liver cells are an important target cells of DV, liver damage in DHF / DSS plays an important role in the pathogenesis of: 1) can be detected in liver tissue DV antigens, and can be separated into infectious virus ; 2) DV can also infect cultured HepG2, HuH-7, HA22T, Hep3B, PLC and other human hepatoma cell lines, culture supernatant can be detected in the mature viral particles; 3) DHF / DSS patients often appear swollen liver large, autopsy shows liver steatosis, Kupffer cell hypertrophy. Elevated serum transaminase levels, thrombin content decreased, the amplitude change and the degree of liver damage and bleeding, shock is closely related. According to the literature, DV can directly infect liver cells, causing liver damage, in other words, the liver may be an important target organ of DV. However, in the past quite a long time, scholars of DV infection coagulation system, endothelial cells and inflammatory cytokine changes done more research, compared to liver cell injury suffered less of a concern, such as energy found that viral replication in liver cells had an impact on what aspects, find a host cell key molecules involved in viral replication, not only for DV infection is important to clarify the mechanism, but also for further study antiviral lay an important foundation. Under physiological conditions, reactive oxygen species (reactive oxygen species, ROS) in the body to maintain a favorable harmless low level of participation in a variety of life processes. When the body adverse conditions, ROS increase in the body and cause tissue oxidative damage in the pathological process is called oxidative stress (Oxidative Stress, OS). Recent studies have shown that the cellular redox state (redox state) of balanced participation of viral replication and pathogenesis. Virus due to lack of energy metabolism system and the necessary enzymes and can not survive independently, strictly dependent on the host cell. Into the cells using cell anabolic macromolecular synthesis system for viruses, thus disrupting the host cell's own metabolic and physiological function, leading to cellular redox balance is broken. Cells through the generation of reduced glutathione (reduced glutathione, GSH), superoxide dismutase (SOD), thioredoxin, touch antioxidant enzyme molecule, keep it to restore state. GSH three cysteine-containing eukaryotic cells the most important aspects of antioxidant. A variety of viruses such as human immunodeficiency virus (human immunodeficiency virus, HIV), hepatitis C virus (hepatitis C virus, HCV) infection in vivo can be changed such as the intracellular redox state, so that it is pro-oxidant status (pro-oxidant ), the OS. Virus can increase intracellular oxidant content, or inhibition of synthesis of antioxidants affect the host cell oxidation balance the performance of the oxidation levels, decreased antioxidant capacity. Peterhans and other first discovered in 1979 by RNA viruses infect host cells, phagocytic cells can produce ROS. They used Sendai virus (Sendai Virus, SV) spleen cells of infected mice, the first confirmed viral infection that causes cells to produce ROS. In 1987 they found influenza viruses and paramyxoviruses vitro activation of monocytes and polymorphonuclear leukocytes produce ROS. HIV infection, the cytoplasm of lung epithelial cells in peripheral blood lymphocytes and the leachate decreased levels of antioxidants, contain high levels of GSH, T lymphocytes are selectively lost. Hennet et al found that influenza A virus infection in mice lungs antioxidants such as GSH and vitamins C and E, has a significant degree of decline. This shows that the virus is closely related to oxidative stress. In addition, there is another report of type 1 herpes simplex virus (herpessimplex virus-1, HSV-1), HCV, etc. These viral infection can decrease the intracellular GSH. Virus infection studies suggest that the redox state of the change is the result of GSH depletion, different viruses infect different cell depletion of GSH, duration and induce different mechanisms. Parainfluenza virus in epithelial cells induced lesions, GSH levels dramatically. The HIV-infected human macrophages with only after the establishment of chronic infection can observe significant decrease in antioxidant levels. Viral infection caused by cellular pro-oxidant state, the virus may be in the process of interaction with the host cell, triggering some factors, and ultimately lead to changes in the intracellular redox state, thereby inducing apoptosis or cell proliferation and other pathological processes. The study suggests that viral infection of host cells may be involved in oxidative stress. Further pathophysiological conditions GSH metabolism study will help people to understand the mechanisms of disease, as well as treatment of the disease to provide new ideas. At present, for DV infection host cell redox status changes rarely reported. Recently, some scholars have been reported in patients infected with DV vivo redox state change. It suggests acute viral infection and other similar, DV infection effect of direct damage to the liver may be affected by the redox state of the liver cells caused. 10 in the DV protein, E protein is the structure of the surface protein in DV, the major envelope protein of viral particles. E gene in virus particles in infected cells play an important role in the process, E protein may have some host cell surface receptor ligand. E protein adsorption of proteins as a virus, the virus enters the cell in the process play a key role. So, E protein on the host cell redox state and what impact? View of the above background, this study aimed to detect dengue virus type 2 (DV2) infection and stable expression of the E protein of human hepatoma cell line HepG2 intracellular GSH changes, and drug treatment changes in intracellular GSH levels after the virus titer changes reflect DV2 infection caused by host cellular redox state changes and their impact on viral infection, preliminary study DV2 infection and intracellular redox state relations . Expected results of this study for further elucidate DHF / DSS pathogenesis, DV pathogenesis and the further design of novel targeted antiviral drugs provide a theoretical basis. The main results and conclusions are as follows: 1. DV2 infection intracellular GSH levels in HepG2 impact DV2 infection group added to MOI = 10 infected HepG2 cells, mock-infected group joined 56 ℃ 30min inactivated virus solution, placed in the same conditions 37 ℃, the initial infection is denoted by 0, the infection 10min, 20min, 30min, 40min, 60min/1h, 2h, 6h, 12h, 24h, 48h (after 5 time points after adsorption 1h, replace the virus maintenance medium ), the cells were washed with PBS, trypsinized, remove cells. After four rapid freezing and thawing, centrifuged supernatant for GSH determination. Take the same amount of cells were sonicated for measuring cellular protein concentration. It was found that virus infection 10min, 20min, 30min, 40min, 60min/1h, HepG2 intracellular GSH levels and mock-infected group, a downward trend, in which the most significant decline in 30min, was 16.82 ± 0.86nmol/mg (n = 5), and mock-infected group 23.14 ± 1.41nmol/mg (n = 5) and infected group compared to other time points were significantly different (P lt; 0.01). Virus infection 2h, 6h, 12h, 24h, 48h, HepG2 cellular GSH levels and mock-infected group, continued to show a downward trend, which 2h, 24h was more evident when, respectively, 29.51 ± 3.16nmol/mg (n = 5), and 17.75 ± 3.32nmol/mg (n = 5), the corresponding time points the mock-infected group 35.45 ± 3.55nmol/mg (n = 5), and 22.91 ± 4.15nmol/mg (n = 5), and infection compared to other time points, significant difference (P lt; 0.05), followed by a gradual recovery to 48h, and mock-infected group had no significant difference (P gt; 0.05). 30min supernatant after infection GSH content was 47.86 ± 3.00nmol/ml (n = 4), compared with mock-infected group increased 33.09%, and there is a significant difference (P lt; 0.05), while the mock-infected group and the virus stock is no significant difference (P gt; 0.05). These results suggest that the HepG2 cells can be infected with DV2 intracellular GSH levels decline, changing the redox state of the host cell, and presented a step change, presumably with viral replication processes. Combining the same time points after infection extracellular GSH levels increased and reported in the literature, suggesting that DV2 infection 30min cellular GSH levels decrease rapidly DV2 infection may be due to increased permeability of cell membranes, GSH caused the leakage. 2. DV2 E protein on the host of intracellular GSH levels built first stable expression of E protein in HepG2 cells pRe-E/HepG2, and by PCR, restriction enzyme digestion, nucleotide sequencing and indirect immunofluorescence were identified and test, it was confirmed that the cell E protein. While building a stably transfected cell lines pRe-E/HepG2 empty vector as a control. Then measured pRe-E/HepG2 intracellular GSH levels. Results: pRe-E/HepG2 intracellular GSH levels and pRe/HepG2 cell control group, showed a downward trend. pRe-E/HepG2 cellular GSH average concentration of 25.61 ± 2.23nmol/mg (n = 4), pRe/HepG2 intracellular GSH average concentration of 33.38 ± 1.07nmol/mg (n = 4), and pRe/HepG2 cell comparison, pRe-E/HepG2 intracellular GSH decreased by 24.3% (P lt; 0.01). Logarithmic growth phase pRe-E/HepG2 and pRe/HepG2 cell culture supernatant 0.5ml GSH concentration was measured and found pRe-E/HepG2 GSH in cell supernatant average 36.72 ± 1.40nmol/ml (n = 4 ), pRe/HepG2 cell supernatant GSH content was 57.41 ± 2.00nmol/ml (n = 4), which is significantly lower than the former, the difference was significantly (P lt; 0.05), the average decline in the former than the latter up 36.35 percent. The above results show that stable expression of the E protein pRe-E/HepG2 stable transfected cell plasmid pRe/HepG2 cells, intracellular GSH concentrations were significantly decreased, suggesting that E protein expression in a host cell can be changed not only within the cell redox state, but also outside the host cells decreased GSH levels in a cell DV2 infection induces changes in the redox state of the process, E proteins may play an important role. 3. BSO and exogenous GSH treatment on the basis DV2 infection in MTT results and according to the literature, select the BSO treatment concentration of 0.2mM and 1mM, processing of exogenous GSH concentration of 10mM and 20mM. In the case of non-infectious virus, the culture supernatant was added to 0.2mM, 1mM BSO treatment 18h, allow intracellular GSH content by the blank control group 24.53 ± 2.59nmol/mg (n = 5), respectively, decreased to 14.29 ± 1.48nmol / mg (n = 5), 14.05 ± 1.93nmol/mg (n = 5) (P lt; 0.05), and the two concentrations decrease was no significant difference (P gt; 0.05), the average rate of 42.24% (n = 5). BSO treatment groups in DV2 infection, infection before 18h respectively 0.2mM, 1mM BSO pretreatment of HepG2 cells, and then use DV2 to MOI = 1 infected HepG2 cells and to maintain the concentration to infection BSO 24h, blank control group without drug treatment. 24h after infection, viral supernatant charged, viral titer was determined by plaque test (PFU / ml). The results showed that 0.2mM and 1mM BSO treatment group than the control group titers were increased by 119.79% (n = 5) and 127.51% (n = 5), compared with the control group is significantly different (P lt; 0.05), but BSO treatment group two concentrations rise virus titer was no significant difference (P gt; 0.05). In the case of non-infectious virus, the culture supernatant was added to 10mM, 20mM GSH solution compared with the control group, intracellular GSH levels were not significantly different (P gt; 0.05); DV2 infection in GSH treatment group, starting from infection beginning to infect supernatant 24h maintain intracellular GSH concentrations were 10mM, 20mM, blank control group without drug treatment. 24h after infection, viral supernatant charged, virus titer (PFU / ml). The results showed that compared with the control group in terms of, 10mM and 20mM GSH treated virus titer decreased 40.24% (n = 5) and 56.62% (n = 5), two concentrations of GSH treatment group and the control group were significantly different ( P lt; 0.05), and the virus titer 20mM GSH treatment group decreased more significantly (P lt; 0.05); These results show not only the cell DV2 can cause changes in the redox state, the intracellular redox state can in turn DV2 affect the replication and proliferation. These results elucidate the pathogenesis of DV and the further design of targeted antiviral provides preliminary experimental evidence.
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