2.0 MATERIALS AND METHODS 2.1 Cell culture techniques All solutions and media for cell culture were made with sterile ultrapure water (Milipore, USA) All cell culture and media preparation work was performed under aseptic conditions in a Class II Type A2 Biosafety Cabinet (ESCO Pte Ltd) Ltd Singapore) The cells used in this study were grown in 25 cm2 or 75 cm2 plastic tissue culture flasks (Iwaki Glass, Japan.), and 24-well tissue culture plates (Greiner Bio – One, USA) Cells were either cultured in a humidified 37 °C incubator (Thermo Fischer Scientific, USA.) with % carbon dioxide or a dry incubator (Memmert GmbH, Germany) 2.1.1 Cell lines The cell lines and the type of media used for each cell line used in this study are listed in Table 2-1 All cell lines used are adherent cell types Table 2-1 Cells and media Cell Line Cell Media Baby Hamster RPMI Kidney Cells 21 (BHK), Human Embryonic DMEM Kidney cells FT (293FT) Mosquito cells L-15 derived from Aedes albopictus (C6/36) | Materials and methods Cell Passage level 100 – 190 10 – 75 80 – 120 Origin American Type Culture Collection, USA Invitrogen Cat no R700-07 Kind gift from Emeritus Professor Edwin Westaway, Australia 26 2.1.2 Media and solution for cell culture All culture media were supplemented with either 10 % or % foetal calf serum [FCS (PAA Laboratories GmbH, Austria)] and their formulations can be found in Appendix 1A to C for the respective cell lines The media pH was adjusted to approximately 7.3 with solutions of M sodium hydroxide and M hydrochloric acid (Appendix 1D) 2.1.3 Cultivation and propagation of cell lines Flasks of cells were sub-cultured from confluent 75cm2 flask monolayer at a ratio of : 10 (BHK 21 and 293 FT cells) or : (C6/36 cells) The growth medium (Appendix 1A to C) was first discarded The monolayer was then rinsed with ml of phosphate buffered saline (PBS - Appendix 1E) This was followed by incubation with ml of trypsin (Appendix 1F) The flask was left to incubate at 37 °C for and the cells were dislodged by gentle tapping Appropriate amounts of growth media were added to the cell suspension to deactivate the activity of trypsin Cell aggregates were resuspended by pippetting up and down The suspended cells were then aliquoted into new tissue culture flasks The cells were then incubated at 28 °C for C6/36 cells or 37 °C for all other cell types 2.1.4 Cultivation of cells in 6-well and 24-well tissue culture plate A confluent cell monolayer of BHK or 293FT cells in a 75 cm2 tissue culture flask was used to seed four 24-well or 6-well plate (Greiner Bio-One, St Louis, USA) The cell monolayer was treated as previously described (Section 2.1.3) to produce a single | Materials and methods 27 cell suspension The cell suspension was then made up to a final volume of 10 ml using appropriate cell culture growth medium Aliquots of ml of cell suspension were then diluted in 24 ml or 30 ml of cell culture growth medium Apporximately x 105 cells or x 106 cells was then transferred into each of the 24 wells or wells, respectively The plates were then incubated at 37 °C in a humidified incubator (Thermo Fisher Scientific, Massachusetts, USA) with % carbon dioxide The monolayers were confluent in 48 hr and ready for use 2.1.4.1 Cultivation of cells on cover slips Individual glass cover slips were aseptically placed in 24-well tissue culture plate A confluent monolayer of cells from a 75 cm2 tissue culture flask was used to seed the wells as described in Section 2.1.4 The plates were then incubated at 37 °C in a humidified incubator supplemented with % carbon dioxide until they were about 80 % to 90 % confluent 2.2 Infection of cells and purification of virus 2.2.1 Viruses The virus used in this study is the Sarafend strain of West Nile virus It was a kind gift from Emeritus Professor Edwin Westaway The virus was propagated in either BHK or C6/36 cells | Materials and methods 28 2.2 Infection of cell monolayers for virus propagation A confluent monolayer of cells was used for infection The cell culture supernatant was discarded and the monolayer was rinsed with ml of PBS (Appendix 1E) Either ml or ml of virus suspension were used to infect cells in 75 cm2 or 175 cm2 tissue culture flask, respectively The flask was incubated at 37 °C for hr and rocked every 15 to ensure even infection After hr, unabsorbed virus was washed off with ml of appropriate maintenance medium (Appendix 2A and 2B) and an appropriate volume of the same medium was added to the flask Subsequently, the infected BHK or C6/36 cell culture flask was either incubated at 37 °C in a humidified incubator supplemented with % carbon dioxide or at 28 °C in a dry incubator, respectively 2.2.3 Preparation of virus pool Infection was carried out as described in Section 2.2.2 The virus was harvested when cytopathic effects were pronounced, usually 24 hr post infection (p.i.) To obtain extracellular virus, the infected cell culture supernatant was removed from the flask and spun at 3000 x g for 10 to remove cellular debris Intracellular virus was obtained by harvesting the monolayer of cell The cells were detached from the flask with an appropriate amount of trypsin (Appendix 1F) Trypsin was neutralize with an equal maintenance medium The resulting cell suspension was sedimented at 800 x g for 10 and followed by cycles of freeze-thawed action The cell debris were removed by spinning at 3000 x g for 15 Approximately 500 µl of infected supernatant (extracellular virus) or clarified cell lysate (intracellular virus) was aliquoted into sterile | Materials and methods 29 cryovials (Nalgene, USA) and immediately snapped frozen in -80 °C liquid ethanol The frozen cryovials (Nalge Nunc International, Roskilde, Denmark) were then stored in a 80 °C freezer 2.2.4 Concentration and purification of virus Supernatant containing virus was prepared as described in Section 2.2.3 Concentration of virus was achieved by placing the supernatant into a Vivaspin 100000 MW centricon filtration device (Satroius, Germany) and spinning at 3000 x g until the desired volume is attained The partially purified virus is either snap frozen in -80 °C liquid ethanol or further purified by density gradient Density gradient is performed with the OptiPrep density gradient medium (AxisShield, Norway) The medium was diluted with 1M Tris-HCl buffer, pH7.4 (Appendix 2C) to obtain a range of concentrations (20 % - 50 %) A ml continuous density gradient was prepared by layering different concentrations (20 %, 30 %, 40 % and 50 %) of ml each of the OptiPrep medium on top of one another starting with the most concentrated solution at the bottom The virus supernatant was layered on top of the gradient and spun at 35000 rpm (Beckman L8-ultracentrifuge, USA) for 16 hr at °C in a SW 55 rotor (slow acceleration, no brakes) After the spin, 1.5 ml of the medium was removed from the bottom of the tube The next ml of medium was extracted from the bottom and diluted 1:1 One ml of the diluted extract was layered over ml of 15% OptiPrep medium as a cushion The layered cushion was spun at 35000 rpm in a SW 55 rotor for hr at °C After the spin, the supernatant was removed and the virus pellet was resuspended in Tris-HCl buffer, pH 7.4 | Materials and methods 30 2.2.5 Plaque assay BHK cells were subcultured and grown in 24-well plates as described in Section 2.1.4 Ten-fold serial dilutions of the virus sample were prepared in virus diluent down to 10-8 (Appendix 2D) Aliquots of 100 µl from each dilution were transferred in triplicates onto confluent cell monolayers in the wells The plate was rocked every 15 for hr at 37 °C with % carbon dioxide to ensure even distribution of virus inocula The diluted virus inocula were removed, and the cell monolayers were washed once with PBS (Appendix 1E) One ml of overlay medium (Appendix 2E) was pipetted into each well The trays were incubated at 37 °C in a humidified % carbon dioxide incubator Plates were incubated for two days before the overlay medium was removed for staining The plaques were visualized by staining the monolayer with 0.5 % crystal violet in a 25 % formaldehyde solution (Appendix 2F) for at least two hr at room temperature on orbital shaker The crystal violet solution was removed for proper hazardous chemical disposal and the plate was washed under a running tap to remove residual dye Plaques were counted and the titre was calculated as PFU per ml of supernatant (PFU/ml) 2.2.6 Extraction of virus RNA Viral RNA extraction was carried out using QIAmp® Viral RNA extraction kit (Qiagen, USA) Virus pool for RNA extraction was obtained at described in Section 2.2.3 Extraction of viral RNA was performed by adding the kit’s lysis buffer to the virus and subsequently loading the lysate into a spin column RNA was isolated from the supernatant by centrifugation RNA bound to the column’s membrane was then eluted with 35 µl of DEPC-treated water (Appendix 3A) | Materials and methods 31 2.3 Molecular techniques 2.3.1 Cloning vectors, expression vectors and infectious clones constructs The following Table (Table 2-2) shows the list of vectors and constructs used in this study Table 2-2 List of genes and regions cloned Name P28-His-C pCMVmyc-C Gene or regions cloned Amino-terminal Histidine-tagged mature capsid protein Amino-terminal cMyctagged mature capsid protein p1.3-transfer The first 1.3kb of the WNV genome pWTIC WNV infectious clone Vector backbone Pet28 A (Novagen) pCMV-myc (Clontech) PBR322 (Promega) PBR322 (Promega) Purpose Expression of C protein in bacteria cells Expression of C protein in bacteria cells A carrier vector for the mutagenesis of the C protein Production of WNV virus All the above clones were constructed previously in the laboratory except for P28-His-C which was constructed specifically for this project Myc-C was constructed by Bhuvanakantham Raghavan while p1.3-transfer and pWTIC was constructed previously (Li et al., 2005) 2.3.2 List of primers Table 2-3 shows a list of the names of the primers used and its corresponding purposes The sequences, of these primers can be found in Appendix 3B | Materials and methods 32 Table 2-3 List of the names of primers used and its purpose No 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Primer Name His-C F His-C- R Sense RNA UTR F Sense RNA UTR R Sense RNA UTR F Sense RNA UTR R Sense RNA UTR+C F Sense RNA UTR+C R A-sense RNA UTR F A-sense RNA UTR R A-sense RNA UTR F A-sense RNA UTR R A-sense RNA UTR+C F A-sense RNA UTR+C R RNA 1F RNA 1R RNA 2F RNA 2R RNA 3F RNA 3R RNA 4F RNA 4R RNA 5F RNA 5R RNA 6F RNA 6R RNA 7F RNA 7R RNA 8F RNA 8R RNA 9F RNA 9R RNA 10F RNA 10R RNA 11F RNA 11R RNA 12F RNA 12R A-sense RNA 1F A-sense RNA 1R A-sense RNA 12R | Materials and methods Purpose Cloning of C protein Synthesize sense 3’ UTR viral RNA Synthesize sense 5’ UTR viral RNA Synthesize sense 5’ UTR+C viral RNA Synthesize anti-sense 3’ UTR viral RNA Synthesize anti-sense 5’ UTR viral RNA Synthesize anti-sense 5’ UTR+C viral RNA Synthesize fragment 1- 1017 of viral RNA Synthesize fragment 960-1974 of viral RNA Synthesize fragment 1920-2934 of viral RNA Synthesize fragment 2880-3894 of viral RNA Synthesize fragment 3830-4839 of viral RNA Synthesize fragment 4790-5804 of viral RNA Synthesize fragment 5758-6772 of viral RNA Synthesize fragment 6728-7742 of viral RNA Synthesize fragment 7698-8702 of viral RNA Synthesize fragment 8655-9669 of viral RNA Synthesize fragment 9613-10617 of viral RNA Synthesize fragment 10053-11057 of viral RNA Synthesize ant-sense fragment 1- 1017 of viral RNA Synthesize anti-sense fragment 10053-11057 of 33 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 A-sense RNA 12R RNA RT Rev RNA RT Rev RNA RT Rev RNA RT Rev RNA RT Rev RNA RT Rev RNA RT Rev RNA RT Rev RNA RT Rev RNA RT 10 Rev RNA RT 11 Rev A-sense RNA RT Rev A-sense RNA RT 12 Rev Seq C F Seq C R viral RNA Reverse primers for real-time polymerase chain reaction for the above generated RNA fragments Forward and reverse sequencing primers for capsid protein All primers were synthesized either by Proligo Singapore Pte Ltd or Sigma-Aldrich The primers are partially purified through desalting procedures performed by the manufacturers 2.3.3 Bacteria strains The One Shot DH5α Escherichia Coli (E.Coli) competent cells (Invitrogen, USA) were used for the transformation and propagation of cloning vectors BL2-CodonPlus competent E.coli cells (Stratagene, USA) were used for the expression of proteins All the bacteria cells were grown in either Luria-Bertani (LB) broth (Appendix 4A) or LB agar supplemented with the appropriate antibiotic (Appendix 4B) | Materials and methods 34 2.3.4 2.3.4.1 Agarose electrophoresis RNA agarose gel electrophoresis To cast a 1.5 % RNA agarose gel, 1.5 g of agarose (1st Base, Singapore) was mixed with 74 ml of deionised water and brought to a boil in a microwave When the molten agarose cooled, 10 ml of 10 x MOPS buffer (Appendix 5A) and 8.75 ml of 37 % formaldehyde was added to give final concentration of x MOPS and 2.2 M of formaldehyde, respectively The molten agarose was poured into a gel casting tray and the combs inserted When the gel had solidified, it was transferred into the gel electrophoresis tank filled with x MOPS buffer and 2.2 M formaldehyde RNA samples were prepared by adding an appropriate amount of RNA loading buffer (Ambion, USA) and µl of 10 mg/ml ethidium bromide was added into the sample to aid visualization after electrophoresis The sample was heated to 65 °C for 10 immediately prior to loading Approximately µl of RNA marker (Ambion, USA) was added in another well to indicate the relative size of the RNA The sample was electrophorized at 120 volts for 40 mins The DNA bands were then visualize under UV light (UV transilluminator, Vilber Lourmat, UK) and captured with ChemiGenius (Syngene, UK) 2.3.4.2 DNA agarose gel electrophoresis To cast a 1.2 % DNA agarose gel, 2.4 g of agarose (1st Base, Singapore) was mixed with 200 ml x TBE buffer (Appendix 5B) in a 500 ml conical flask and heated to a boil in a microwave The flask of molten agarose was cooled and µl of 10 mg/ml of ethidium bromide solution (Sigma, USA) was added and mixed The molten agarose was then poured into a gel casting tray and the gel combs were inserted The solidified gel | Materials and methods 35 M Supernatant Pellet 100 50 40 30 25 20 His-C 15 10 Figure 3-3 Solubility of His-C protein Transformed bacteria culture was induced with 1.0 M IPTG and harvested hr post induction The cells were sonicated in lysis buffer and the cell debris was separated from the supernatant via centrifugation Both the supernatant and the insoluble cell debris were subjected to SDS-PAGE analysis to ascertain if His-C protein is found predominantly in the supernatant (Lane 2) or the cell debris pellet (Lane 3) The black arrowhead indicates the over-expressed His-C protein The marker is in Lane | Results 62 3.2.3 Purification of histidine-tagged capsid (C) protein The soluble fraction of the cell lysate was passed through the nickel ion infused IMAC membrane adsorber for purification However, it was observed that His-C was not captured on the membrane; instead it was detected in the flow-through (Fig 3-4A, Lane 1) Therefore, cobalt ion was chosen to capture His-C protein Similarly, the cobalt ion infused IMAC membrane was also unable to capture the protein (Fig 3-4B) Because the capsid protein is highly positively charged at neutral pH, it may inhibit the binding of the protein to the membrane Theoretically, raising the pH of the solution the protein is in would reduce the positive charge of the protein and therefore improve the binding efficiency of the protein Indeed, when the pH of the cell lysate was raised to 8.5 from the initial 7.8 with M NaOH, His-C protein was observed to be excluded from the initial flow-through and was only eluted out with M imidazole (Fig 3-4C) Next the maximum and minimum concentration of imidazole needed to wash the membrane and elute His-C protein from the membrane was determined, respectively The results showed that the highest concentration of imidazole used to wash the membrane without eluting His-C protein, was 750 mM while the minimum concentration needed to elute His-C was 1000 mM (Fig 3-5) | Results 63 Figure 3-4 Purification of His-C protein (A) Purification with Nickel ions Clarified supernatant of bacteria total cell lysate was allowed to pass through the IMAC adsorber infused with Nickel ions and the resulting flow through (FT, Lane 1) was collected The membrane was then washed with 50 mM (W1, Lane 2), 100 mM (W2, Lane 3), and 150 mM (W3, Lane 4) of imidazole Subsequently, any bound protein was eluted with 500 mM (E1, Lane 5) and 1000 mM (E2, Lane 6) of imidazole The molecular marker is in Lane The black arrowhead indicates the presence His-C protein in the FT No protein is eluted in Lanes and (B) Purification with Cobalt ions The purification process with a cobalt ion infused IMAC membrane is similar to (A) Similar to (A) no proteins are eluted in Lanes and The black arrowhead indicated the expected size of the HisC protein (C) Purification of with Cobalt ions after raising the pH of the cell lysate supernatant to 8.5 The pH clarified supernatant of bacteria total cell lysate was raised to 8.5 before it was allowed to pass through the Cobalt ion infused IMAC membrane As in (A), the cell lysate went through the same washes and elution Bound protein is eluted in Lane and The black arrowhead indicates the purified His-C protein | Results 64 A FT W1 W2 W3 E1 E2 M FT W1 W2 W3 E1 E2 M His-C B Expected size of His-C protein FT W1 W2 W3 E1 E2 M C His-C | Results 65 FT W1 W2 W3 W4 W5 E1 E2 E3 M 150 75 50 37 25 20 15 His-C 10 10 Figure 3-5 Optimized purification profile of His-C protein Clarified supernatant of bacteria total cell lysate was allowed to pass through the IMAC adsorber infused with Cobalt ions and the resulting flow through (FT, Lane 1) was collected The membrane was then washed with 200 mM (W1, Lane 2), 300 mM (W2, Lane 2), 500 mM (W3, Lane 4), 750 mM (W4 Lane 5) and 1000 mM (W5, Lane 6) of imidazole Subsequently, any bound protein was eluted with 1500 mM (E1, Lane 7), 2000 mM (E2, Lane 8) and 2500 mM (E4, Lane 9) of imidazole Smaller molecular weight bands (indicated by black arrow) observed were most likely degradation of the main product The molecular marker is in Lane 10 The black arrowhead indicates the expected size of the purified His-C | Results 66 Therefore, the final optimized protocol was to wash the membrane with 750 mM of imidazole and elute the bound His-C protein with 1500 mM of imidazole Most of the His-C protein was eluted with x 20 ml washes of 1500 mM of imidazole (Fig 3.6A) The identity of the induced protein was confirmed with both Western blot (Fig 3-6B) and mass spectrometry (Appendix 12) Finally all purified His-C was pooled together, concentrated down to ml (Fig 3-7A) and quantitated with Bradford assay using bovine serum albumin as standards (Fig 3-7B) The yield was approximately 250 ng/ml A B M FT E1 E2 E3 E4 E5 E6 E7 E8 M P CL 150 75 50 37 25 20 His-C 15 10 10 Figure 3-6 Purification, elution and confirmation of the identity of the His-C protein (A) Clarified supernatant of bacteria total cell lysate was allowed to pass through the IMAC adsorber infused with Cobalt ions and the resulting flow through (FT, Lane 1) was collected The membrane was washed with 750 mM of imidazole and the bound protein was eluted with x 20 ml washes of 1500 mM of imidazole (E1-E8, Lanes 3-10) The marker is in Lane Most of the protein is eluted by the 8th wash (B) Confirmation of the identifty of the eluted protein A sample from the E3 eluate (P, Lane 2) and the total bacteria cell lysate from uninduced cells (CL, Lane 3) were subjected to Western blot analysis with anti-His antibodies The marker is in Lane Arrowhead indicates His-C protein | Results 67 B A OD His-C [BSA] ng/ml Figure 3-7 Quantification of His-C protein (A) An SDS-PAGE analysis of the purified His-C protein after concentration The marker is in Lane while the His-C protein (arrowhead) is in Lane (B) Bradford assay A bovine serum albumin standard curve was created to measure the concentration of the His-C protein The equation of the best fit like is indicated above The OD reading of the concentrated His-C protein is about 0.48, which corresponds to a concentration of 250 ng/ml | Results 68 3.3 Synthesis and biotinylation of viral RNA Viral RNA was synthesized as described in Sections 2.6.1.1 and 2.6.1.2 The positive strand and negative strand of the 5’ UTR , 5’ UTR + capsid and 3’ UTR of the viral RNA were checked for integrity before biotinylation (Fig 3-8) Initial attempts at detection of biotinylation through a dot blot assayed failed because an unsuitable blocking reagent was used, resulting in over-developed blots (Fig 3-9A) This was overcome with a commercial blocking reagent (Fig 3-9B) The dot blot assay was able to detect up to ng of labelled RNA (Fig 3-9B) M 3’UTR 5’UTR+C 5’UTR kb 0.5 kb Figure 3-8 RNA gel electrophoresis RNA representing the 3’UTR (Lane 2), 5’UTR+C (Lane 3) and 5’ UTR (Lane 4) were synthesized and analyzed its for size and integrity by gel electrophoresis RNA marker is in Lane and the molecular weight of the smallest RNA are indicated on the left | Results 69 A B 16 Amount of RNA (ng) 5’ UTR 5’ UTR + C 3’ UTR Figure 3-9 Detection of biotinylated RNA (A) Biotinylated RNA was dot blotted onto a PVDF membrane (red circles) and detected with streptavidin conjugated to alkaline phosphatase The blot was developed with alkaline phosphatase substrate (B) As in (A), biotinylated 5’ UTR, 5’UTR + C and 3’ UTR RNA was serially diluted times and dot blotted on to a PVDF membrane The blot was then blocked with a suitable blocking reagent to prevent over development of the blot as seen in (A) and probed with streptavidin-conjugated to alkaline phosphatase (black dots) | Results 70 3.4 Northwestern blot assay The Northwestern blot was developed to study capsid and RNA interaction This is usually done with radio-labelled RNA, but since biotinylated RNA was used here, optimization was needed The assay was initially developed with a colorimetric assay but was found to be unsatisfactory since only the 3’UTR of the viral RNA was detected (Fig 3-10A) when as much as 40 ng of C protein was used Hence, it was decided to use chemiluminesence to detect for bound RNA on the blot This turned out to be better and much more sensitive since interaction was detected between His-C protein and all species of the RNA even with as little as 1.25 ng of protein (Fig 3-10B) | Results 71 B A 150 5’ UTR 5’ UTR +C 3’UTR 5’ UTR 5’ UTR +C 3’UTR 75 50 37 20 15 10 Figure 3-10 Northwestern blot assay (A) Purified His-C protein was resolved by SDSPAGE and blotted onto nitrocellulose membrane for probing with biotin-labelled viral RNA representing the 5’UTR (Lane 2), 5’ UTR + Capsid [(C) Lane 4] and 3’ UTR (Lane 6) of the West Nile virus genome The markers are in Lane 1, and The blot was developed with a colorimetric assay and only the interaction with 3’UTR is detected (Lane 6) (B) Similar to (A), biotin -labelled viral RNA representing the 5’UTR (Lane 1), 5’ UTR + Capsid [(C) Lane 2] and 3’ UTR (Lane 3) of the West Nile Virus genome were used to probe for purified His-C protein blotted on nitrocellulose membrane The labelled RNA were detected with a chemiluminescent assay and developed with an x-ray film developer Arrowheads indicate positive interaction | Results 72 3.5 RNA pull-down assay To complement the study of Northwestern blot assay in the study of C and RNA interaction, a RNA pull-down assay was developed In order to determine the minimum amount of RNA needed to pull-down C protein, the amount of C protein and streptavidin beads was kept constant but a range of concentration was used for the RNA and it was found that as a little as 1.25 ng of RNA could be used to pull down C protein (Fig 311A) Next, the minimum concentration of streptavidin beads needed to pull down the C – RNA complex was determined The complex was allowed to form and a range of streptavadin concentration was used It was found that at least 20 µg was needed to pull down detectable amounts of C protein (Fig 3-11B) For subsequent experiments we would be using at least 20 µg of streptavidin beads and at least 0.5 ng of RNA since the RNA can be synthesized in abundance A B RNA Concentration (ng/ml) 20 15 10 0.5 2.5 1.25 Streptavidin beads (µg) 0.5 70 60 50 40 30 20 10 Figure 3-11 RNA pull-down with streptavidin magnetic beads (A) His-C protein was allowed to complex with 20 ng/ml to 0.5 ng/ml of viral 3’ UTR RNA (Lanes 1-7) and the complex was pulled down with 70 µg of streptavidin beads (B) His-C protein was allowed to complex with 0.5 ng/ml of viral 3’UTR RNA and the complex is pulled down with a range of concentration of the streptavidin beads They range from 70 µg to 10 µg (Lanes 1-7) Arrowheads indicate positive pulldown results | Results 73 3.6 Production and validation of anti-C antibodies with His-C protein Twenty-five µg of His-C protein was injected into the mouse every fortnight and the mouse was sacrificed weeks after the first injection The serum was harvested and tested An immunoblot was performed to test the sensitivity of the serum to detect His-C protein derived from infected cell lysate and C protein peptide fragments (Fig 3-12) In addition to immunoblot assays, the antibodies were tested on infected cells and visualized with fluorescence microscopy (Fig 3-13) | Results 74 Infected BHK cell lysate Mock infected BHK cell lysate His-C A B Peptide Fragment 1-23 18-45 41-64 61-84 79-105 100-123 Dimeric His-C Monomeric His-C Figure 3-12 Validation of serum from mouse inoculated with C protein via immunoblot (A) His-C protein (Lane 1), mock-infected BHK cell lysate (Lane 2) and infected-BHK cell lysate (Lane 3) were blotted onto a nitrocellulose membrane after SDS-PAGE The membrane was incubated with inoculated mouse serum diluted 1:100 in % milk in TBST The antibody bound to the membrane was then detected using anti-mouse antibody conjugated to HRP Finally the blot was developed with a chemiluminescent assay The antibody was able to detect His-C protein (arrow) as well as the dimeric form (small arrow) of the C protein in infected cells (B) Overlapping peptide fragments spanning the entire C protein (Lanes 1-6) were synthesized and dot blotted onto a nitrocellulose membrane The membrane was incubated with mouse serum diluted 1:100 in % milk in TBST and the anti-C antibody bound to the membrane was then detected using anti-mouse antibody conjugated to HRP Finally the blot was developed with a chemiluminescent assay Only peptides 1-5 react with the antibody since peptide was not expressed in the His-C protein | Results 75 hr post-infection Mock-infected A B 24 hr post-infection CC Figure 3-13 Immuno-staining of WNV C protein in infected cells with anti-C antibodies BHK cells is mock-infected (A) or infected with WNV virus and fixed at hr (B) or 24 hr (C) post-infection The cells were then probed with anti-C antibodies to test the specificity and sensitivity of the antibodies Anti-mouse alexafluor 594 secondary antibodies were then used to stain the cells The nuclei of the cells are stained with DAPI C proteins are stained predominately in the nuclei at hr post-infection (B) while the C proteins are stained predominately in the cytoplasm at 24 hr post-infection (C) The scale bars are shown on the bottom right corner | Results 76 ... A-sense RNA UTR+C R RNA 1F RNA 1R RNA 2F RNA 2R RNA 3F RNA 3R RNA 4F RNA 4R RNA 5F RNA 5R RNA 6F RNA 6R RNA 7F RNA 7R RNA 8F RNA 8R RNA 9F RNA 9R RNA 10F RNA 10R RNA 11F RNA 11R RNA 12F RNA 12R A-sense... Appendix 3B | Materials and methods 32 Table 2- 3 List of the names of primers used and its purpose No 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39... 5758-67 72 of viral RNA Synthesize fragment 6 728 -77 42 of viral RNA Synthesize fragment 7698-87 02 of viral RNA Synthesize fragment 8655-9669 of viral RNA Synthesize fragment 9613-10617 of viral RNA