University of Leuven Laboratory of Virology and Experimental Chemotherapy Department of Microbiology and Immunology Rega Institute

A compiled Practical Report

By: Abel Abera Negash Year I IPMB student Supervisor: S.J.F.Kaptein, PhD May 2010

1. Introduction Dengue virus (DENV), a species in the genus Flavivirus causes an acute febrile illness in humans with symptoms ranging

from clinically inapparent to severe fatal

hemorrhagic disease. There is an incubation period of 5–8 days, and the symptoms last about 10 days with severe headaches, retro-ocular pain, back and limb pains, nausea and vomiting. Often there is a scarletiniform or maculopapular rash. There is no specific treatment, but analgesics containing acetaminophen can be used to relieve pain. The virus is only transmitted by the bite of an infected mosquito vector, and cannot be spread from person to person. Following infection, humans and nonhuman primates usually develop a high level of viremia lasting about 5 days, and if a competent mosquito vector takes a blood meal during this viremic phase, it becomes infective after 8–12 days and capable of transmitting the virus. Replication of dengue virus occurs in HeLa cells with CPE and in suckling mouse brain. The virus can be adapted to eggs and other cell types. Adapted experimentally to mice, it causes flaccid paralysis of the limbs. Most nonhuman primates have an inapparent infection following experimental inoculation. Currently, control of dengue fever relies upon control of the mosquito vector, and despite much developmental research, no vaccine is available. See also dengue viruses 1–4 . dandy fever virus; polka fever virus. Gubler DJ (2006)[1] Novartis Found Symp 277, 3 dengue viruses 1–4 (DENV-1–4). Four serotypes of Dengue virus in the genus Flavivirus which together with Kedougou virus form the dengue virus serogroup. Double diffusion tests reveal a common antigen and specific antigens. Type 1 occurs in South-East Asia from India to Japan and Hawaii, with temporary spread to Greece, South Africa, and Australia. The virus is not endemic in Japan except for a transient outbreak in the 1940s. Type 2 occurs in SouthEast Asia, central America, and the Caribbean. Types 3 and 4 occur in Thailand and the Philippines. Hemorrhagic fever with dengue shock syndrome probably results from infection with one type in persons immune to

another. An antigen–antibody reaction occurs in the tissues and results in increased vascular permeability and leakage of plasma, but the molecular basis of the pathological process is not well understood. No vaccines are currently licensed, but considerable progress has been made toward the development of a tetravalent live attenuated vaccine. Control is presently aimed at the principal vector species, Aedes aegypti.[1,2,3]

2. Cell culture Cell culture is the most common method of propagation of animal viruses. The cells will be grown in a chemically defined and buffered medium that is optimal for cell growth. For Dengue virus Vero cells and BHK (Baby hamster kidney)cells are used. Derived from the kidney of an African green monkey (Cercopithecus aethiops) in the 1960s, Vero cells are one of the most common mammalian continuous cell lines used in research. Since they are anchorage dependent they cannot be used in suspension. Vero cells usually take 2-3 passages to reach their regular growth rate, and this should be taken into account if planning to use the cells for experiments, infections, etc. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) containing 5% fetal bovine serum (FBS) (Invitrogen) and 100U/ml penicillinstreptomycin.

2.1.Subculturing The cell monolayer is observed under microscope to examine the general condition of the cells before each subculturing. If the cells appear in good shape and free of contamination trypsinization follows. The previously spent medium is aspirated and 10ml of PBS is used to wash off the remaining serum which would inhibit the enzyme activity of the Trypsin-EDTA. The wash solution is then aspirated and 3ml of TrypsinEDTA is added. Trypsin is a porcine pancreas-derived enzyme that is commonly used for the dissociation of cells from the culture substrate. The concentration of trypsin necessary to achieve this can vary and is dependent on a variety of factors. EDTA on the other hand is a chelating agent with gentle dissociative properties that acts to increase enzymatic efficiency by neutralizing calcium and magnesium ions.

The falsk is then incubated at 370c for 5 minutes until the cells detach. Gently tapping the bottom of the flask also accelerates the process if any cells are left undetached. When cells detach 4ml of growth medium is added. The whole of the flask surface is washed by Pipetting up and down in order to recover all the cells and break clumps. The suspension will then be centrifuged and the supernatant aspirated. The cells are then resuspended with 1 or 2ml medium and a 1:5 dilution is pipette into a new 75 cm2 flask. The medium is then added to make a final volume of 20ml and the cells are incubated at 370c.[4]

3.Dengue virus Replicons Subgenomic replicons of positive-stranded RNA viruses, which contain all the nonstructural proteins required for amplifying themselves but lack some or all of the structural proteins, have been shown to be useful tools to study viral replication in the absence of virion assembly and maturation [8–11]. Because of the unique features including high level of expression, no DNA intermediate, exclusively cytoplasmic replication, and relative ease of construction, subgenomic replicons offered great potential as vectors for gene expression and vaccine development. The replicon could be detected via a luciferase-reporter. Firefly luciferase is a 62,000 dalton protein which is active as a monomer and does not require subsequent processing for its activity. When luciferin is added to a sample containing luciferase , there is an immediate light flash that reaches peak intensity at 0.3–0.5 seconds, and then decays rapidly. This rapid exponential decay is caused by the reaction product, oxyluciferin, which inhibits luciferase activity. [5,6,7]

Fig 1. Dengeu Virus replicon

4.The Antivaral Assay Despite the considerable impact of the pathogenic flaviviruses on human health, there is no effective antiviral therapy. However, several antiviral compounds such as ribavirin and interferon (IFN) have proved to be active in vitro against the replication of YFV, DENV and JEV. [8] In the laboratory of Virology and experimental chemotherapy, Rega Institute, University of Leuven, a whole library of Antiviral compounds against DENV-2 New Guinea have been screened and some interesting hits have been found. The ongoing work therefore encompasses trying to find an antiviral compound which will best fit for transformation into an invivo trial surpassing the invitro parameters. The primary screening is semi-automatic and involves seeding cells in a 96 well microplate with 7000 cells/well. Next day cells are infected with DENV-2 New Guinea in the presence or absence of a serial dilution. In this procedure virus-infected cultures are incubated with antiviral compounds for a period sufficient to permit virus replication and then assayed for the presence of new progeny virus by titration on separate monolayer cultures.

4.1.Standard Assay: Inhibition of Viral Cytopathic Effect (CPE) This test, run in 96 well flat-bottomed microplates, will be used for the initial antiviral evaluation of all new test compounds. In this CPE inhibition test, four dilutions of each test compound (e.g. 50, 10, 2, 1 µg/ml) will be added to 3 cups containing the cellmonolayer; within 5 min, the virus is then added and the plate sealed, incubated at 37°C and CPE read Read out is done at day 8 post infection by fixing and staining the cells using 1% methylene blue solution. A known positive control drug, Ribavarin is evaluated in parallel with test drugs in each test.

Fig. 2. Setup of the cell control, virus control and test dilutions on a 96 well plate during primary screening.

4.2.Evaluation-Verification/Profiling assay Follow-up testing with compounds found active in initial screening tests are run in the same manner except 8 1:2 dilutions of each compound are used. This Involves seeding cells at 7000 cells/well. Next day the cells will be infected with DENV-2 new guinea in the presence of serial dilution of the compounds. The data are expressed as 50% effective concentrations (EC50).

Fig 3. Setup of the test dilutions, virus and cell control during profiling assay

4.3.Virus yield experiment For compounds considered active by CPE inhibition ,effect on reduction of virus yield will be examined. Cells are seeded at 50000 cells/well. Next day, cells are infected with DENV-2 New Guinea and theVirus is incubated for 1-2 hours Virus is removed and cells are washed 3× using assay medium (2%FCS/MEM) Compounds are added and diluted 1:2 As in the initial tests, a known active drug is run in parallel as a positive control.

4.4.Cytotoxicity Assay Along with virus yield experiment it is essential to perform a cytotoxicity assay. This will help to assess the cytotoxicity of the compounds as the best interest is to find hit compounds with the least or no cytotoxicity possible. In the CPE inhibition tests, two wells of uninfected cells treated with each concentration of test compound will be run in parallel with the infected, treated wells. At the time CPE is determined microscopically, the toxicity control cells will also be examined microscopically for any changes in cell appearance compared to normal control cells run in the same plate. These changes are given a designation of numbers ranging from 0 to 5. For further analysis though MTS/PMS assay is performed. This assay is performed in 96 well plate and uses the novel tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) and the electron coupling reagent, phenazine methosulfate (PMS). MTS is chemically reduced by cells into formazan, which is soluble in tissue culture medium (8). The measurement of the absorbance of the formazan can be carried out using 96 well microplates at 492nm. The assay measures dehydrogenase enzyme activity found in metabolically active cells. Since the production of formazan is proportional to the number of living cells, the intensity of the produced color is a good indication of the viability of the cells. Typically, in MTS-based assays, toxicity is determined using the dose-response curve to

determine EC50, the concentration of the test substance required to reduce the light absorbance capacity of exposed cell cultures by 50%. However, IC50 values can also be determined by generating a standard curve consisting of a specified range of cell dilutions, because a 50% reduction in absorbance may not equate with a 50% reduction in cellular viability. [9]

5.Taq Man assay The Supernatant that has been harvested at day 4 post infection during the virus yield experiment is used for Real time Quantitative PCR. The real-time RT-PCR assay is a one step assay system used to quantitate viral RNA and using primer pairs and probes that are specific to each dengue serotype. The use of a fluorescent probe enables the detection of the reaction products in real time, in a specialized PCR machine, without the need for electrophoresis. Many real-time RT-PCR assays have been developed employing TaqMan or SYBR Green technologies. The TaqMan real-time PCR is highly specific due to the sequence-specific hybridization of the probe. SYBR green real-time RT-PCR has the advantage of simplicity in primer design and uses universal RT-PCR protocols but is theoretically less specific.

6.References 3.Alvarez M et al Dengue hemorrhagic fever caused by sequential dengue 1-3 virus infections (2006) Am J Trop Med Hyg 75 , 1.Gubler DJ Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 11 , 480,1998 2. Gubler DJ and Meltzer M Dengue: an escalating problem , Adv Virus,1999 4. A. J. Can Virus Culture: A practical approach , 2002, Oxford University press 5. Blight K.J., Kolykhalov A.A. and Rice C.M., Efficient initiation of HCV RNA replication in cell culture. Science 290: 1972–1974, 2000. 6. Khromykh A.A., Replicon-based vectors of positive strand RNA viruses. Curr. Opin. Mol. Ther. 2: 555–569, 2000. 7. Khromykh A.A., Sedlak P.L. and Westaway E.G., Cis- and trans-acting elements in flavivirus RNA replication. J. 8. Leyssen et al . Perspectives for the Treatment of Infections with Flaviviridae, Clinical Microbiology,. 2000, 9. www.promega.com/enotes/applications/ap0017_tabs.htm