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CHAPTER0 4 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT Forvirus SALE OR Distributionreplication Cyclesnot for SALE OR DISTRIBUTION CHAPTER0 4 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FORVirus SALE OR DISTRIBUTIONReplication CyclesNOT FOR SALE OR DISTRIBUTION © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Some human and animal virusesNOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION are grown in tissue culture cells in vitro. This researcher is seeding dishes of tissue culture cell monolayers.© Jones The monolayers & Bartlett are Learning, LLC © Jones & Bartlett Learning, LLC grownNOT in a CO2 FOR incubator SALE before OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION the cells are infected with a laboratory strain of virus. Viruses that can be grown in cell cultures © Jones &can Bartlett be well characterized Learning, in the LLC © Jones & Bartlett Learning, LLC NOT FORresearch SALE laboratory. OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION o u t l i n e © Jones & BartlettIn the struggleLearning, LLC4.1 One-Step Growth© Curves Jones & Bartlett4.3 Learning, The Error-Prone LLC RNA “ Polymerases: Genetic Diversity NOT FORfor SALE survival, OR DISTRIBUTION the 4.2 Key Steps of theNOT Viral FOR SALE OR DISTRIBUTION Replication Cycle 4.4 Targets for Antiviral Therapies fittest win out at 1. Attachment (Adsorption) RNA Virus Mutagens: A New Class the expense of 2. Penetration (Entry) of Antiviral Drugs? their rivals © Jones & Bartlett 3. Uncoating Learning, (Disassembly LLC Virus© Fil Jonese 4-1: How& Bartlett Are Cellular Learning, LLC NOT FOR SALEand OR Localization) DISTRIBUTION ReceptorsNOT Used FOR for ViralSALE OR DISTRIBUTION because they Attachment ­Discovered? 4. Types of Viral Genomes and succeed in adapt- Their Replication Virus File 4-2: Unraveling the Life Cycle of Mimivirus ing themselves 5. Assembly © Jones & Bartlett Learning, LLC © Jones & Vi rusBartlett File 4-3 Learning,: Stamping DownLLC 6. Maturation Flu Viruses bestNOT to theirFOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 7. Release Refresher: Molecular Biology environment.” Charles Darwin © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 67 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. CASE0STUDY:0 HUMAN0METAPNEUMOVIRUS © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION The campus day care was recently closed during the samples are sent to diagnostic laboratories, and a peak of the winter flu season because many of the lesser percentage in adults. young children were sick with a lower respiratory Both hMPV and RSV cause upper and lower tract infection. An email announcement was sent respiratory tract infections associated with serious to all students,© Jones faculty, &and Bartlett staff at the Learning, college that LLC illness in the young, immunosuppressed,© Jones & Bartlett elderly, Learning, LLC stated the closureNOT wasFOR due SALE to a metapneumovirus OR DISTRIBUTION and chronically ill. CommonNOT FOR symptoms SALE includeOR DISTRIBUTION outbreak. The announcement briefed the campus cough, fever, wheezing or exacerbation of asthma, community with information about human meta- and rhinorrhea (runny nose). It can produce se- pneumonoviruses (hMPVs). vere enough symptoms to cause intensive care The announcement stated that hMPV was a admission and ventilator support. Healthy adults © Jonesnewly & identified Bartlett respiratory Learning, tract LLC pathogen discov- can get© aJones mild form & Bartlettof the disease Learning, that is character- LLC NOTered FOR in theSALE Netherlands OR DISTRIBUTION in 2001. New tests confirm ized byNOT a cough, FOR hoarseness,SALE OR congestion,DISTRIBUTION runny that it is one of the most significant and common nose, and sore throat. viral infections in humans. It is clinically indistin- Members of the campus day care staff were guishable from a viral relative known as respiratory doing their best to limit the epidemic spread of the syncytial virus (RSV). Both RSV and hMPV infec- hMPV outbreak at the center. Primary care physi- © Jones & Bartletttions Learning, occur during LLC the winter. hMPV may account© Jones cians& Bartlett need up-to-date Learning, knowledge LLC and heightened NOT FOR SALE ORfor 2% DISTRIBUTION to 12% or more of previously unexplainedNOT FORawareness SALE OR to recognizeDISTRIBUTION this new viral disease in pediatric lower respiratory infections for which patients. n Chapter© Jones2, you learned & Bartlett that viruses Learning, are de- LLC animals were difficult© to Jones work with & Bartlettand expensive Learning, LLC pendentNOT upon hostFOR cells SALE for their OR reproduction, DISTRIBUTION to maintain. AnotherNOT drawback FOR was SALE that animals OR DISTRIBUTION Iyet to do so viruses must overcome certain cel- were not very permissive to infection with human lular constraints. Only those viruses that have viruses due to the species barrier and the animal been able to adapt to their hosts have been able to immune response. exist in nature. The previous chapter focused on Enders, Weller, and Robbins won the 1954 © Jonestaxonomy & Bartlett and structure Learning, of viruses LLC and in sum- Nobel© Prize Jones in Physiology & Bartlett or Medicine Learning, for theLLC cul- NOTmary FOR stated SALE that OR several DISTRIBUTION different virus families tivationNOT of FORpoliovirus SALE in nonnervousOR DISTRIBUTION tissue cul- cause similar viral disease syndromes. tures (human embryonic skin and muscle cells). This chapter focuses on experiments such as Their observations and procedures used to grow one-step growth curves, which are used to viruses in vitro contributed to the refinement of study virus–host interactions. These studies have tissue culture techniques and played a monumental © Jones & Bartlettpro Learning, vided information LLC about the events that ©occur Jones role& Bartlett in the development Learning, of LLC vaccines against polio- NOT FOR SALE ORat each DISTRIBUTION step of the infection cycle (attachmentNOT, FORvir SALEus in the OR 1950s DISTRIBUTION (Salk vaccine) and 1960s (Sabin penetration, uncoating, replication, assembly, vaccine). maturation, and release), including the intricate One-step or single-step growth curve ex- details of the strategies that animal and human periments are used to study a single replication viruses use to express and replicate their diverse cycle of viruses. Max Delbruck developed the one- genomes. © Jones & Bartlett Learning, LLC step growth curve experiment© Jones &while Bartlett using Learning, an LLC NOT FOR SALE OR DISTRIBUTIONEscherichia coli-T4 bacterialNOT system, FOR which SALE gave OR faster DISTRIBUTION experimental results than did traditional methods. 4.1 One-Step Growth With the advent of cell culture systems, these ex- periments were carried out with viruses that infect Curves tissue culture cells. These experiments are per- © Jones & Bartlett Learning, LLC formed© Jonesin special & cell Bartlett culture facilities.Learning, LLC NOTVirologists FOR SALE could OR not DISTRIBUTION study animal and human Briefly,NOT FOR monolayers SALE OR(or cellDISTRIBUTION suspensions in viruses well in the laboratory before tissue cul- liquid medium) of tissue culture cells such as mon- ture methods were developed by John F. Enders, key kidney cells are allowed to adhere and form Thomas H. Weller, and Frederic C. Robbins in the monolayers on the bottom of plastic dishes. The late 1940s. Before their work, viruses were injected monolayers of cells are subsequently infected with © Jones & Bartlettinto Learning, animals and LLC tissues were analyzed for© theJones the& Bartlett virus of choice.Learning, They LLCare infected at a high NOT FOR SALE ORpathological DISTRIBUTION signs of viral infection. ExperimentalNOT FORmultiplicity SALE OR ofDISTRIBUTION infection (MOI) to ensure that 68 ChapteR 4 Virus0Replication0Cycles 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. General Procedure: One-Step Growth Curves FiGure 4-1 The diagram briefly outlines the steps involved © Jones & Bartlett Learning, LLC © Jones & inBartlett performing Learning,one-step growth LLCexperiments. Step 5 includes NOT FOR SALE OR DISTRIBUTIONStep 1: NOT FOR SALEa photograph OR of DISTRIBUTION viral plaques (clearings where the virus Infect monolayers of tissue destroyed the cell monolayer). The plaque assay is a culture cells (using a vertical quantitative assay used to determine the number of viruses laminar flow biosafety hood) present in a given sample. The results of these assays can be and allow the infection to used to generate a one-step growth curve for a particular virus. proceed in a CO2 incubator. For more details about virological methods see Chapter 5, Laboratory Diagnosis of Viral Diseases and Working with © Jones & Bartlett Learning, LLC Viruses in the Research Laboratory.© Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Step 2: Monitor experiments via CO2 incubator. inverted microscope. Step 3:© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Collect infected cell lysates at variousNOT time pointsFOR after SALE infection. OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Step 4: Perform serial dilutions on infected cell lysates and
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