| Dengue virus belongs to the Flaviviridae of flaviviruses, diseases caused by dengue virus include:dengue fever (DF), dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). Severe dengue (dengue hemorrhagic fever and dengue shock syndrome) was first recognized in the1950s during dengue epidemics in the Philippines and Thailand. According to the WHO reported in November2012, the incidence of dengue has grown dramatically around the world in recent decades. Over2.5billion people, about half of the world’s population was now at risk from dengue. The disease was now endemic in more than100countries in Africa, the Americas, the Eastern Mediterranean, South-east Asia and the Western Pacific. Cases across the Americas, South-east Asia and Western Pacific had exceeded1.2million cases in2008and over2.3million in2010(based on official data submitted by Member States). Recently the number of reported cases has continued to increase. Not only the incidence and mortality of dengue fever had grown dramatically around the world in recent decades, but also the disease spread to new areas, explosive outbreaks were occurring. In2012, Dengue fever was classified the propagation velocity of the fastest vector-borne viral disease by WHO, it had the possibility of epidemics in the world. The world needs to change coping style, and implementation of sustainability precautions. Dengue fever had become a major international public health concern.The increases in the incidence and distribution of DEN fever and DEN hemorrhagic fever were due to several reasons. No effective vaccines are available for DENV. Vector control programs had been curtailed or discontinued, and pesticide-resistant insects had emerged. Increasing urbanization in the tropics and rapid world travel had greatly extended the range of A. aegypti. So to develop new tools is an emergency work. Such as developing a vaccine against dengue/severe dengue,genetic manipulation of vector mosquitoes to render them incompetent for virus transmission. The genetics of vector competence were poorly understood. An alternative genetic mechanism to alter host susceptibility to virus infection was the phenomenon known as pathogen-derived resistance (PDR). The DPR was found in plants first, synthesis and accumulation of tobacco mosaic virus coat protein in transgenic plants mediated intracellular immunity to tobacco mosaic virus infection. This phenomenon was named the RNA interference (RNAi). RNAi is a cellular process induced by double-stranded RNA(dsRNA) capable of producing the RNase-mediated degradation of specific mRNA. RNAi was found to be widespread in the biosphere, from low prokaryotes, fungi, invertebrates, mammals discovered this phenomenon. The RNAi antiviral effect has been found in a variety of insect vector, Ronald P. van Rij found that RNAi pathway in Drosophila melanogaster Ago-2deficient strain viral RNA showed high levels of accumulated in vivo. Vargas knockout the expression of enzyme AGO-2, Dcr-2and R2D2of RNAi pathway, can increase the replication of dengue virus type2in Aedes aegypti in vivo. Researchers had begun to use RNAi interference technology modification or transformation of the mosquitoes, which reduced carrying or not carrying the virus. Recently, several studies have described the antiviral efficacy of chemical-, plasmid-, or virus-vectored small dsRNAs in both cell cultures and mouse models infected with various flaviviruses such as West Nile virus (WNV), Japanese encephalitis virus(JEV), and yellow fever virus (YFV).DENV infection and transmission could be suppressed in mosquitoes transgenically engineered to produce DENV-targeted small interfering RNA (siRNA). Adeno-associated virus (AAV) vectors containing short hairpin RNA (shRNA) targeted to the CS1region inhibited DENV-2infections in mammalian cell.RNAi was expected to become a new strategy for the prevention and control of dengue virus infection. Dengue viruses contain a single-stranded, positive-sense RNA of approximately11Kb. The virion RNA contains a single long open reading frame encoding three structural proteins and sevennon-structural proteins, arranged in the order5’-C-prM-E-NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5-3’. prM protein was further cleaved by host cell proteases late in infection to produce the nonglycosylated M protein found in the mature virion and involve the viral assembly, maturation and maintenance of infectivity. So prM was selected as trigger RNAi effector. Successfully mediated RNAi would also need stable and efficient expression systems. Plasmid vector had the following advantages:different selectable marker, long-term stability of gene expression and low cost of large scale genetic screening.In our research, we used a vector which highly expressed in a variety of mammalian cells and cloned the inverted-repeat RNA derived from the genome of DENV-2into it, which yielded plasmid pcDNA-irRNA. The plasmid pcDNA-irRNA was transfected into BHK-21cell and the anti-viral effects analyzed by Semi-quantitative PCR and real-time PCR. We provided a theoretical basis for new strategies for prevention and control of dengue virus infection.Objective:1. Clone the inverted-repeat RNA derived from the genome of DENV-2into pcDNA3.1(+) which highly expressed in a variety of mammalian cells, which yielded plasmid pcDNA-irRNA.2. Construct the recombinant plasmid pcDNA-eGFP and transfected BHK-21cells to observe transfection efficiency.3. To evaluate the anti-viral effects of the plasmid which designed to transcribe an inverted-repeat RNA derived from the genome of dengue virus type-2capable of forming dsRNA.Methods:1. Suckling mice were inoculated with live DENV-2in the brain and the total RNA was extracted from the brain of the infected mice. 2. the membrane glycoprotein precursor M(prM) gene fragments were amplified by RT-PCR then subcloned into Xho I/EcoR I of the pcDNA3.1(+) in antisense orientation,which yielded plasmid pcDNA-asprM.3. Another DENV-2prM sequences were amplified by RT-PCR and subcloned into pMD18-T-vector in sense orientation,which yielded plasmid pMD18-T-prM.4. pcDNA-irRNA was constructed by inserting the prM fragment in sense orientation isolated from pMD18-T-prM into the Nhe I/Kpn I of the pcDNA-asprM.5. PCR amplify the eGFP fragment and cloned into pcDNA3.1(+) construct the expression plasmid pcDNA-eGFP.6. Plasmid pcDNA-eGFP was transfected into BHK-21to observe the transfection efficiency.7. Dengue virus inoculated from the suckling mice is used to infected BHK-21and collect the culture supernatants.8. Detect the dengue virus infection of BHK-21cells by Immunofluorescence.9. Passage dengue virus in BHK-21cells and determine TCID50.10. The recombinant plasmid pcDNA-irRNA and pcDNA were transfected into BHK-21cell.11. Transfected cells were challenged by dengue virus type2, total RNA of the cells were collected at different days after infection.12. The mRNA expression of NS3of the experimental group (pcDNA-irRNA, transfected and DENV-2challenged),the positive group (DENV-2challenged) and blank control group(BHK-21cells transfected with pcDNA3.1(+) and DENV-2challenged) analyzed by Semi-quantitative PCR.13. The viral load in different time points of the experimental group and the positive control group were detected by real-time PCR.14. The mRNA expression of NS1in experimental group and the positive control group were analyzed by real-time PCR.Results: 1. Dengue virus type-2was successfully collected from the Suckling mice in the brain and the total RNA was extracted from the brain of the infected mice.2. The prM gene fragments from the dengue virus type-2were cloned and the length of the gene fragment is600bp. which was subsequently confirmed by DNA sequencing analysis and agarose gel electrophoresis.3. Recombined plasmid pMD18-T-prM, pcDNA-asprM and pcDNA-irRNA which contains inverted-repeated RNA were successfully constructed.4. It was confirmed by DNA sequencing that the eGFP cDNA sequence in recombinant plasmid pcDNA-eGFP was in accordance with the EGFP cDNA sequence in plasmid pEGFP-N(Gene blank accession:#U55762). pcDNA-eGFP plasmid was Successfully constructed.5. BHK-21cells were infected with the recombinant plasmid pcDNA-eGFP and the transfection efficiency of pcDNA-eGFP was detected by the expression of enhanced fluorescent protein. The transfection efficiency of pcDNA-eGFP in24h,48h,72h and96h were36.3%、45.0%、31.6%'31.3%.6. Dengue virus type2was successfully passaged in BHK-21cells and undiluted virus titer was106.48TCID50/0.1ml.7. Transfection with the plasmid pcDNA-irRNA caused a reduction of NS3mRNA expression level by28%in BHK-21cells following a96-h challenge with DENV-2as compared to the cells without plasmid transfection (positive control), which is detected by RT-PCR and analyzed by Glyko BandScan software.8. Ten-fold dilutions of the pMD18-T-NS1plasmid DNA and samples were tested by the established FQ-PCR assay to evaluate the sensitivity of the system, our established FQ-PCR method using dilution series of pMD18-T-NS1plasmid DNA to calculate the end-point sensitivity of each assay. The results indicated that the established FQ-PCR is a high sensitivity, specificity, simplicity, and reproducibility PCR assay and this method is suitable for DENV-2detection.9. The FQ-PCR result showed that viral copies in pcDNA-irRNA-transfected cells was1.44-fold lower than those in the positive control cells following a72-h virus challenge and26.7%in96hpi. 10. The mRNA expression levels of NS1were also significantly lower in the transfected cells at96h after viral challenge (P<0.05) as shown by real-time quantitative PCR.ConclusionThe plasmid pcDNA-irRNA which contained irRNA was successfully constructed, The observations suggested that inverted-repeat RNA derived from the genome of DENV-2was consistent with RNA silencing as the mechanism of resistance to DEN-2in BHK-21cells, which provided a theoretical basis for the study of dengue virus gene vaccine. |
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