Analysis of RSV Virus - Essay Example

FILE In infants, the RSV virus remains a major epidemiological factor to infections of the respiratory tract. However, the virus is equally manifested in adults, especially those predisposed to the pulmonary disease in its chronic phase, or persons with impaired immune systems. In attempts to combat the infection, there has been development of RSV inhibiting molecules. Such molecules, however, are not angle to fully control multiplication of the coral species. Im addition, attempts to develop a working anti-RSV vaccine have been fruitless, though development of the immune-specific monoclonal Palivizumab antibody. This antibody has proved essential in management of the virus development in succeptible children. To date, Ribavirin stands as the only accredited drug against the circus. Nevertheless, the applicability of this sophisticated drug has been subject to cost and toxicity constrains thus limiting its use. As a consequence, there remains an urgent need for research ventures aimed at developing more effective anti-RSV treatments. To fuse with the host cell, the virus uses glycoprotein F; therefore, this knowledge could be used in drug targeting to develop anti-protein F medications. Through research ventures, the first strain of the virus was harvested from the chimps respiratory channel, and has since been widely associated with infants and newly born babies. Notably though, the virus lacks age specificity with its incedences being noted among adult humans. Currently, it is estimated that the disease causes approximately 34 million infections of the lower respiratory tract among children. Out of these, an approximate 10% often require hospitalization to recover. Sadly, up to 7% of the hospitalised children end up losing their battle against the virus, with fatality rates in developed states accounting for just 1% of the total. While world health systems are continuously laying foundations in attempts to develop an ideal vaccine and medication against the infection, this has faced many hurdles. In the end, the world remains stuck with Palivizumab as the only humanized antibody compliant to the guidelines of the Food and Drugs Authority. This artificially articulated MAb targets the virus by identifying its antigenic F-protein and mounting immunological response against the virus. On the other hand, the virus can be managed through transcription arrest in this RNA-virus, an approach that has been exploited in the designing of Ribavirin. However, such RNA targeting drugs are highly costly to produce,, while others are very toxic. Therefore, there stands a need to rapidly develop ideal treatment mechanisms against the disease, a critical concept that this paper critically analyses. Structurally, the negatively sensed RSV virus is neither is founded on a single stranded RNA as its genetic material. The virus belongs to the Paramyxoviridae family, and possesses a non-segmented body. It has membrane glycoproteins G and F, both of which are integral in host-cell fusion. The virus membrane is equally highly glycsylated at its G residues as opposed to the low degree of glycosylation in F sequence. This renders sequence G a less potent immunological target compared to F. Consequently, drug-search tests are less directed towards glycoprotein G. Biochemically, the F glycoprotein is a type exoprotein which traverses the membrane, and is formed from a pre-cursor that has an approximate Mass of 67kD. The proteins amino terminal acts as a signaling end upon cleavage. The cleavage and consequent activation of the protein is carried out by specialized proteolytic enzymes, a process that occurs at the trans face of the golgi apparatus. Following action of the pro teases on the To precussor, two polipeptides joined through sulphur bridges are formed. These two include the F2 polypeptide formed from the amino terminal and the F1 polypeptide formed from the carboxyl terminal. Thence, a 27 unit polypeptide referred to as pep27 is released by the golgi. The various ends of the pep27 have fur in cleavage sites called HRA, HRB and HRC and the cytoplasmic domain- Viruses 2013, 5 213 Figure 1. A representation of the RSV glycoprotein F and its fusion to target host cells. (A) diagrammatic elaboration of the action of protein cleaving enzymes on precursor molecule forming two subunits; the F1 and F2 units. The figure outlines the SP, HRC, HRA, HRB, TM and CP domains of the virus. (B) Shows an RSV representation indicating glycoprotein F fusing to a target surface. The structure of F changes following fusion of protein G to G-receptors. The conformation of protein F then shifts into HRA helix which is long. The ends of Tue FP act as receptor binding sites. The association forms trimmers which further associate to form the time Ric HRB which associate further with the HRA forming a denser unit; the 6-HB. The dense unit promotes close association between the virus membrane and the target cell membrane. F-protein occurs in a variety of forms. Of the forms, fusion form of the protein is trimeric, crystalline and metastable. Uncleaved F protein has tendency to realign to form the folded form through a wide sequence of changes in conformation. In recent times, pre-triggered soluble forms of the protein have been isolated through deletion of its cytoplasmic and transmembrane units. Similarly, sF protein has been shown to exist in single states free of protein-protein interactions. Under manipulative effects of mild buffers though, the sF protein is able to form aggregates which occur as rosettes. Through research ventures, the sF domains have been exploited for possible use as probes in studies aimed at unveiling the biochemical mechanisms involved in activation of the F protein of the RSV. Through such studies, as revealed in X-ray scans, it is apparent that strategic interaction between the HRA and the HRB lead to formation of coiled structural alignments. Through X-ray crystal analysis tools, it has further been noted that three HRA forms are involved in the process, binding to 3 domains of anti-parallel HRB domains. Such interaction results in the formation of the helical bundle unit. For quite a long time, the atomicity of the virus has been problematic. However, latest solutions, developed through post-fusion analyses, have pointed towards existence of two-head domains, 1 and 2, which conform into a crown-like structure. On the other hand, concurrent studies have indicated that interactions between HRC and HRA units are integral in the development of the neck region, just below the crown. To date, it is a general belief that infections with the virus commence with the successful attachment of the virus to the neck region. Such attachment involves interaction with the glycosaminoglycan (GAGs) components, such as chondroitin sulfate B and heparin sulfate, of the viral membrane. However, developing evidences point towards partial dependence of the virus on G-protein-mediated GAG binding, thus raising more questions on the possible ideal mechanisms. As such, the analogy of ICAM and nucleolin use is constantly gaining ground in elaborating in vitro infection with the RSV. This is further supported by observations made on the ability of recombinant RSV to infect hosts despite their lack of G-proteins. However, the efficiencies of such infections have been observed to be lower than in cases where the protein is present. In this regard, it is also proposed that the F-protein can equally be integral in mediating both membrane fusion and viral attachments. Therefore, it remains widely acceptable that the functionality of the F-protein in the RSV is comparable to the gp41 observed in the HIV-1 virus. Indeed, the F-protein mediated envelop fusion is purported to be highly pH sensitive. Following successful binding of the G and F domains to their respective receptor sites, conformational transitions are initiated. This leads to exposure of target membranes. Thence, the HRA and HRA domains begin to interact, forming the stable hexameric core accompanied by apposition of the membrane. Eventually, the interactive sequences terminate in formation of the fusion pores through which the siRNA from the virus are released into the host genomic setup. The siRNA result into endosome formation, a process that results in clathrin-mediated endocytosis. Therefore, it is apparent, in this sense, that F- protein plays a lead role in the process that leads to fusion between the viral particles and the endocytic vesicles. FILE 2 9/6/14 Lab Book Cell Cultivation The HEK 293T/17 cells grown in the T75 flasks enriched with a medium enriched with Dulbecco’s MEM. This specially designed medium contains glutamax-1, sodium pyruvate, glucose, invitrogen and pyridoxine. The medium also comprises 10% FBS of Latin origin. For selectivity, the medium is based on penicillin antibiotic. It may equally use PBS and TrypLE as alternative selective markers. The medium requires thermal heating to temperatures of 37ºC prior to use. The technique also involves washing of cells with 3cm3 of the PBS after which they are incubated in the presence of TrypLE at temperature ranges, correspondent to normal human body temperatures. This incubation stage is allowed to stand for 4 minutes. Following the incubation, the 7cm3 of the medium is dispensed into the flask. To two T75 flasks, 2 million cells are grown in each flask supplied with 15ml of the freshly prepared culture medium. DAY 1 Seeding a T75 flask It is important to grow the target cells at a population density correspondent to the population in the well plates. To achieve this fete, a comparative assessment of the surface area is made between a 1.75x 10-2m radius plate and a T75 flask. Through this assessment, it is notable that the plates were 0.1284 times relative to the flasks. As such, a single flask was mathematically determined to have the capacity to effectively support 4.6 million HEK cells. The cells were later removed from the flask and counted through dilution techniques using fresh media. The dilution was carried out at a rate of 0.3 million cells for every 1cm3 of the fresh medium. Finally, seeding was conducted on the two flasks using 15.58cm3 of diluted cells. DAY 2 ****TO PREVENT INHIBITION OF TRANSINFECTION, IT IS ADVISABLE TO AVOID USE OF BLUE EPPENDORF TUBES SUPPLIED BY TREFFLAB **** The cells contain plasmid DNA which are essential components in the genetic setup. The plasmid DNA include pFR-Luc and the pcDNA3.1_Gal4/ NFκB Transfections 1 Luc + A2_F_1 2 Gal4 Table 1. transfection constituents Transfection # Reagent 1 2 Serum free media 779 µl 779 µl Fugene 6 23.37 µl 23.37 µl Luc (1 µg/ml) 7.79 µl - Gal4 (1 µg/ml) - 7.79 µl A2_F_1 (1 µg/ml) 7.79 µl - **prior to dispensation, the Fugene is given time to reach room temperature. This is followed by vortex technique which should take in excess of 1 second. ** A medium free of serum is obtained. 1.5ml of the medium is then dispensed in an eppendorf tube followed by careful addition of fugene 6 to the dispensed medium. During the addition of the fugene, care is taken to prevent contact with the plastic surface of the test tubes. Thereafter, the tubes are subjected to vortexing for over one second followed by the incubation procedure which lasts 5 minutes. The incubated components are then enriched through addition of plasmid DNA, a procedure which precedes another moment of vortexing for 1 second, and a further incubation for quarter-hours. After incubation, transfection reagents are added to the components of the flask components. This is facilitated by tilting the flask. After addition of the components, the flask is returned to its original standing position in such a way that the cellular components in the medium are not disturbed. The components af the flask are then incubated overnight at temperatures of 37oC under adequate supply of carbon IV oxide. DAY 3 Compound preparation Various compounds are used in this technique. Such compounds are promptly prepared in such a way that cell growth is enhanced. The compounds are diluted in a round-bottomed flask with the capacity of 96 well plates. The dilution ration adopted is 1:3 to give a dilution curve for a wide variety of concentrations. A blank is equally run to give comparative values with which the various determined curves can be compared. It is notable that all compounds are comprised of 10mM of 100% DMSO. Preparation of curves; A 10mM stock solution was obtained. From the solution, a 4ul preparation was made and dispensed in an eppendorf. To the 4ul, 96ul DMSO was added to make 100ul. 400ul of this preparation was made from which 135ul was transfered into the first well of the first column of the plate which is composed of 96 wells. To each of the other wells, 100ul DMSO was added. 15ul was pipette from the 400uM solution and added into the first column, 1. This was mixed to give a solution with a concentration of 40uM. 50ul of this resultant solution was placed in the second column, 2, followed by thorough mixing. Such transfer was repeated through the remaining columns. Preparation of curves [top] = 500 nM; A 10mM stock solution was obtained. From the solution, a 10ul preparation was made and dispensed in an eppendorf. To the 10ul, 90ul DMSO was added to make a 1mM solution. 400ul of this preparation was made from which 135ul was transfered into the first well of the first column of the plate which is composed of 96 wells. To each of the other wells, 100ul DMSO was added. 15ul was pipette from the 1mM solution and added into the first column, 1. This was mixed to give a solution with a concentration of 100uM. 50ul of this resultant solution was placed in the second column, 2, followed by thorough mixing. Such transfer was repeated through the remaining columns. Preparation of curves [top] = 1 µM; A 10mM stock solution was obtained. From the solution, a 20ul preparation was made and dispensed in an eppendorf. To the 20ul, 80ul DMSO was added to make a 2mM solution. 135ul DMSO was transferred into the first well of the first column of the plates. To each of the other wells, 100ul DMSO was added. 15ul was pipette from the 2mM solution and added into the first column, 1. This was mixed to give a solution with a concentration of 200uM. 50ul of this resultant solution was placed in the second column, 2, followed by thorough mixing. Such transfer was repeated through the remaining columns. Preparation of curves [top] = 3.3 µM; A 10mM stock solution was obtained. From the solution, a 20ul preparation was made and dispensed in an eppendorf. To the 20ul, 80ul DMSO was added to make a 2mM solution. To all the other wells, 100ul DMSO was added. This formed a 0.66mM solution. 50ul of this resultant solution was placed in the second column, 2, followed by thorough mixing. Such transfer was repeated through the remaining columns. Cell preparation This is done through preparation of 5 luciferase plates. The cells are then transfected into the plates as outlined in the above sections. The cells are then counted through dilution techniques using freshly prepared media. The dilution is done using 0.4 million cells per dilution cell. The growth is then analysed and curves reflecting growth rates plotted. Fig 1. Plate layout At body temperature and 5% concentration of carbon IV oxide, the plate components were allowed to stand overnight. Day 4 (24 hr time point) The luciferase assay is essential in this analysis. However, the assay requires several LAAB buffer constituents including: 1. Tricine at a concentration of 20mM. This buffer has a pH of 7.8 and an Mr value of 179.17 2. Magnesium sulphate at a concentration of 10mM and a Mr value of 246.48. This translates to 2.46g of the constituent in per dm3. 3. DTT constituent at a concentration of 10mM and a Mr value of 154.25, translating into a mass of 1.54 in 1 litre. 4. EDTA buffer at a concentration of 1mM and a Mr value of 292.24. This reflects a mass requirement of 0.292g for every litre. To constitute the LAAB buffer, tricine is added to water at a pH of 8.0. Suitable volumes of the remaining constituents are then added, raising the pH to 8.2. The resultant medium is then topped up to appropriate volumes using suitable volumes of water. Prepared buffer is stored under refrigerating conditions with temperatures of -20oC. Lysis buffer This buffer is composed of three main constituents which include: 1. Tris-phosphate; the ideal concentration used is 25mM. Since the compound has a relative molecular mass of 219.13, it translates to a total requirement of 5.47g of tris-phosphate for every litre. 2. Magnesium Chloride; this is required in concentration of 8mM. The compound has a molecular mass of 95.21 translating into a requirement of 0.761g for every one litre. 3. DDT; this third constituent of the buffer is required in concentration of 1mM. It has a molecular mass of 154.25kD hence the total requirement by mass of this compound is 0.154g for every litre. Following assembly and mixing of these three constituents, the pH stands at 7.8. Other constituents added thereafter include 1% Triton X-100 and 15% glycerol. The buffer is then stored at relatively low temperatures of -20oC. **** TO PREVENT OXIDATIVE DEGRATION OF DDT, BOTH LYSIS BUFFER AND THE LAAB BUFFER SHOULD BE THAWED AT 25OC **** Luciferin substrate This is constituted of four main constituents: 1. Tris-HCl; 100mM of this component is required in formulation of the substrate. Since the component has a Mr value of 157.6, it translates into requirement by mass of 15.76g for every litre. This component has a relative pH value of 7.8. 2. Coenzyme A; in constituting the substrate, this component is required in concentrations of 13.5mM. The molecular mass of the component is 318.4 hence a requirement by mass for this constituent is 10.36g for every litre. 3. Luciferin; this is required in concentrations of 23.5mM. The relative mass of this compound is 318.4 hence a mass of 7.48g is required per litre. 4. The final compound is the Adenosine triphosphate; the requirement for this compound is 26.6mM, 14.66g/L by mass. The compound has a relative mass of 551.14. The substrate is highly photosensitive and thermosensitive. As a result, it should be kept away from light at very low temperatures of -80oC. Luciferase Plates Following the discarding of the media into virkon, 100ul PBS was used to wash each of the wells. However, due precautionary measures were observed in aspirating the media and in adding PBS to the wells. This is because the rate at which HEK becomes detached at mild to adverse conditions is rapid. To each well, 20ul of the lysis buffer added and incubated in a shaker for about 300 seconds at 25oC. Luciferin is then added to the LAAB at a dilution rate of X0.02. This generates a working luciferin buffer which is consequently added to the wells at a rate of 100ul per well. This leads to conversion of luciferin to oxyluciferin. The latter is highly luminescent. As such, luminescence is measured immediately. Resazurin Plates To the virkon, media was discarded. 100ul of SFM and 20ul of cell titre were then added to each well. This was followed by incubation of the plates at 37oC in presence of carbon dioxide for 2 hours. Resorufin was then added to the mixture. This compound was reduced to Resazurin, a product that gets excited at wavelengths between 560nm and 590nm. Peak florescent occurs at 590nm and is measurable immediately. Read More