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Bio 329

by: Parneet Notetaker

Bio 329 BIO329

Parneet Notetaker
U of L
Cellular and Molecular Biology
Dr. Paul Himes

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Cellular and Molecular Biology
Dr. Paul Himes
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This 5 page Class Notes was uploaded by Parneet Notetaker on Tuesday March 15, 2016. The Class Notes belongs to BIO329 at University of Louisville taught by Dr. Paul Himes in Spring 2016. Since its upload, it has received 19 views. For similar materials see Cellular and Molecular Biology in Biology at University of Louisville.


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Date Created: 03/15/16
DENV Dengue virus can cause dengue feverdengue shock symptom and dengue hemorrhagic fever in humans 1There are currently no vaccines to fight this virus2The dengue virus is transmitted to humans by female Aedes aegypti mosquitoes while they feed on their blood Current measures to control the spread of dengue include keeping the vector population numbers to a minimum through fogging and other measures include preventing mosquito bites through the use of mosquito repellent and mosquito nets3For the past 50 years much effort has gone into the development of vaccines against DENV but the biggest obstacle of ADE Antibody development enhancement a phenomenon in which the sub neutralizing antibodies themselves help the virus gain entry in some of the cells still stands in the way45 The traditional vaccine approaches included weakened live viruses and plasmids whereas the new approaches focus on transmission blocking vaccinesTBV which aim at breaking the life cycle of the virus within the mosquito which leads to blocking the transmission of the virus by the mosquito to humans6 8 Hence the gap in the knowledge was the effective ways to combat the DENV virus by finding suitable targets for treatment According to results of clinical trials none of the traditional vaccines provide a long lasting protection against DENVThe hypothesis the authors of this paper spend most of the time testing is that all the mosquito genes unregulated during the Infection must have a role in either virus survival or mosquito defense against the infection which suggests that altering gene eXpression would in some way or the other affect virus survival therefore providing important information to design an effective transmission blocking vaccine9 The researchers used comprehensive microarray analyses to find out significant alterations in the range of RNA and mRNA of Aedes aegypti during DENV infection they identified 203 unregulated and 202 down regulated genes10 The researches used siRNA to silence 100 unregulated genes out of the 203 in the cell line Aag 2 of Aedes aegypti and later infected them with DENV72hSilencing 9 of these genes increased cell toxicity beyond measure and silencing 55 of them decreased infection more than 40 when compared to the controlOut of the 55 CRVP39s had a trypsin inhibitor domain which meant that CRVP39s could possibly be a serine protease inhibitor from which they concluded that CRVP and specifically CRPV 379 cysteine rich venom proteinas it was highly up regulated during DENV infection are associated with DENV infection The researchers went on to determine the phenotype of CRV379 during infection with loss of function eXperimentsThey reduced CRVP 379 gene eXpression with the help of RNAi and siRNA The reduced CRVP 379 eXpression reduced DENV infection as a function of time as opposed to the control of GFP siRNA transfected cells10 The researchers transfected pAcCRVP379 vector into Aedes aegypti and later infected it with DENV on the other hand they used a vector eXpressing GFP as control to figure out if increase in CRVP 379 levels increases DENV infection9 After some time they isoltaed the RNA from control and eXperimental cells and found out that increasing CRPV 379 after a certain optimum point does39t increase DENV infection in the cells10After establishing that CRVP 379 was essential in DENV of Aedes aegypti they designed eXperiments to figure out the requirements for DENV infection in live mosquitoesThey used double stranded dsRNA against CRVP 379 coding region and gave it to mosquitoes through intra thoracic injections after 2 4 and 8 days they dissected the mosquitoes and measured the CRVP 379 expression and found that there was almost a complete reduction of eXpression in 70 of the mosquitoes They repeated the same procedure on another set of mosquitoesfed them DENV infected blood and dissected heir midguts and found that complete silencing of gene eXpression almost completely inhibited DENV infection They used GFP dsRNA infected mosquitoes as control After establishing a correlational relationship between CRVP 379 and DENV infectionthe researchers figured the physical role of CRVP 379 Tandem affinity purification assay9 They found that prohibitin a mosquito protein binds to CRVP 379 during DENV infection They used siRNaA to silence the prohibitin gene and found that this greatly decreases DENV infection therefore they established that prohibitin is a DENV receptor and blocking the interaction of the protein with DENV and prohibitin inhibits DENV infectionl 1 This research is important because currently there is no vaccine against DENV and around 2 billion people are at a risk of getting infected by DENV whereas 25000 people die of dengue every year9This research is important because it provides a better target for prevention and treatment of DENV as compared to the traditional vaccines which possibly could transform a mild health condition into a life threatening condition through antibody development enhancementThis research focuses at inhibition of DENV while inside the vector which also reduces the chances of antibody development enhancement This research talks in depth about CRVP 379 gene and the affects of it39s alterations on DENV inhibition which happens to be significantly up regulated during not only DENV but also West Nile Virus and Yellow Fever Virus so information gained regarding CRVP through this research might prove as a scientific break through for an even better vaccine than already known todayThe research increases our knowledge on the topic of DENV pathogenesis in the Aedes aegypti mosquito vector9 The research is not restricted to DENV it provides great deal of information regarding diseases caused by viruses belonging to the genus Flavivirus This research provides insight in techniques or possible approaches that can be used to develop vaccines against viruses belonging to the genus Flavivirus for example the West Nile Virus and the Yellow Fever Viruses have the same significantly up regulated gene as in the case of DENV9 The Zika Virus also belongs to the genus Flavivirus so this research can possibly help design a similar eXperimental setup which might possibly provide answers which the current world needs due to the recent Zika outbreak in many parts of the world The results very closely support the conclusions The overall conclusion is that the CRVP 739 is a possible strong candidate for vaccine target and the results from all the different small subset eXperiments build on one another and support the conclusion as a whole The sub set eXperiments in part are conducted on the mosquito Aag 2 cells and in part on live mosquitoes The eXperiments identify the affects of silencing the CRVP 379 gene and the prohibitin gene on the DENV infectionThis article talks about silencing genes and we talked in class about how genes can be turned on or off with the help of heterochromatin and euchromatin DNAThe mosquito DENV relationship is commensalism and human DENV relation is parasitism which is also something we discussed in class The popular press article conveys the same message as the scholarly journal article but in summary on top of the article suggests that the possible target for transmission blocking vaccine ie CRVP 379 affects the DENV after the Aedes aegypti feeds on the blood of the infected host12 but the article focuses mainly on how the CRVP 379 can be blocked to inhibit DENV so that the infection does not get transferred to humans hence the name transmission blocking vaccine and what the press article states is a small part of the research The article suggests that next step the researchers are taking is based on one of the findings towards the end of the research ie that human blood eXhibits a antibody response to CRVP379 after the mosquito drinks human blood the antibodies remain active for a few hours and possibly help combat the infection for a those few hours9There can be three things that can be done to further this research the first can be introducing the human gene that gives rise to the antibodies in the mosquitoes but this method has some ethical issues possibly eXpensive and this mutant mosquitoes might not be able to survive outside the lab The second approach can be to find a way to prolong the life of the human antibodies in side the mosquito so that they can combat infection and the third approach can be to figure out what factor is causing these antibodies to be ineffective as a whole inside humans because humans still die of DENV every year The next step in line with this research paper should be finding a way to block the CRVP 379 gene or prohibition gene in all mosquitoes so that DENV can be inhibited which like the above idea could be achieved through human blood Instead of trying to catch every mosquito and silencing it39s target genes humans should design a method to feed the mosquitoes the target gene silencing quot agentsquot through their blood so that the infection is inhibited inside the vectors which will possibly lead to prevention of dengue and other diseases The only problem is that the mosquitoes will have already passed the infection to the person who will give them the silencing agents which is good for the second person the mosquito might come in contact with but not so good for the first which brings the discussion back to point that it is important to figure out the various details about the antibodies that work against the CRVP379 proteins Bibliography 1 Gubler DJ 2002 Epidemic denguedengue hemorrhagic fever as a public health social and economic problem in the 21st century Trends Microbiol 10 100 103 pmid11827812 doi 101016sO966842x01022880 2 Guzman MG Halstead SB Artsob H Buchy P Farrar J et al 2010 Dengue a continuing global threat Nat Rev Microbiol 8 S7 16 doi 101038nrmicro2460 pmid21079655 3 van den Berg H Velayudhan R Ebol A Catbagan BH Jr Turingan R et al 2012 Operational efficiency and sustainability of vector control of malaria and dengue descriptive case studies from the Philippines Malar J 11 269 doi 1011861475287511269 pmid22873707 4 Kliks SC Nisalak A Brandt WE Wahl L Burke DS 1989 Antibodydependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever Am J Trop Med Hyg 40 444 451 pmid2712199 5Halstead SB 2003 Neutralization and antibodydependent enhancement of dengue viruses Adv Virus Res 60 421 467 pmid14689700 doi 10101630065352703600114 6 Fink K Shi PY 2014 Live attenuated vaccine the first clinically approved dengue vaccine Expert Rev Vaccines 13 185 188 doi 101586147605842014870888 pmid24350687 7Viar L Dayan GH ArredondoGarcia JL Rivera DM Cunha R et al 2015 Efficacy of a tetravalent dengue vaccine in children in Latin America N Engl J Med 372 113 123 doi 101056NEJMoa1411037 pmid25365753 8 Carter R 2001 Transmission blocking malaria vaccines Vaccine 19 2309 2314 pmid1 1257353 doi 101016sO264410x00005211 scholarly journal article 9LondonoRenteria B Troupin A Conway MJ Vesely D Ledizet M Roundy CM et al Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein PLoS Pathog PLOS Pathogens 201511 10 Colpitts TM Cox J Vanlandingham DL Feitosa FM Cheng G et al 2011 Alterations in the Aedes aegypti transcriptome during infection with West Nile dengue and yellow fever viruses PLoS Pathog 7 e1002189 doi 101371journalppat1002189 pmid21909258 11 Kuadkitkan A Wikan N Fongsaran C Smith DR 2010 Identi cation and characterization of prohibitin as a receptor protein mediating DENV 2 entry into insect cells Virology 406 149 161doi 101016jviro201007015 pmid20674955 popular press article 12interrupting the transmission cycle ScienceDaily ScienceDain 2015 Retrieved httpswwwsciencedaiycomreeases201510151022161108htm


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