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Microbiology and Immunology
Microbiology and Immunology 2500A/B
Cumming/ Damjanovski

Virology Course: viral infectious cycle, viral structure, viral entry, viral pathogenesis, HIV pathogenesis & vaccines Impact for society - Ex. measles virus: most infectious virus, very contagious. Vaccination made disease less prevalent in Canada - Travelling spreads viruses Where can we find viruses? - Viruses infect all living things - Probs eat it daily (insect viruses on vegetables) - Millions in tsp of ocean water—Calicivirus infection of whale (rashes, gastroenteritis) - viral genetic seq integrated into our own genes (~10% DNA is derived from retroviruses) o ex. endogenous retrovirus (HERVs)  incorporated remnants of viruses, likely no longer infectious o ex. exogenous retroviruses (HIV)  carry reverse transcriptase, infectious, originated outside human body - most viruses that infect us don’t cause disease. 3 levels of defense mechanisms (intrinsic, innate immunity, acquired immunity) All viruses are not evil - ex 1. Polydnaviruses, wasps and caterpillars. Wasp genes carried by the polydnavirus virions suppress innate immune response of larva/caterpillar (wasp lays eggs inside caterpillar + virions) allowing development of eggs - virus helps a fungus. Fungus permits plant survival but fungus can’t grow >50°C. virus allows thermotolerance - virus making nicer flowers (broken tulip b/c of potyvirus) Major concepts in virology - in order for viruses to survive they must o package their genome inside a particle, use particle to transfer their genome from host to host, genome contains info to initiate and complete viral infectious cycle, genomes establish themselves in host ensuring long term viral survival. GENOME IS KEY. It orchestrates synthesis of all req elements o 1950s: Established that viral genome is the genetic code - Viral genomes=obligate molecular parasites (intracellular parasites). Can only function after they replicate in host cell - Viruses must make mRNA that can be translated by host ribosomes. Use host protein synthesis machinery to make viral proteins. NO VIRUS CAN TRANSLATE PROTEINS FROM mRNA ON THEIR OWN History of virology - Existed in dinosaur age - Ancient Greeks and Romans documented herpes - Key (first) experiment in understanding fundamental concept: tobacco mosaic virus (TMV). o Crushed TMV infected leaves & filtered. Virus in cell filtrate (bottom of flask) (diff from bacteria). Filtrate couldn’t grow on its own but if rubbed on new leaves it infected - Virus can be filtered through a 0.2 micron filter (not always though) Seeing viruses - 10nm-100nm size, seen through electron microscope. Mimivirus is an exception (can be seen through light microscope and doesn’t pass through 0.2 micron filter therefore never in filtrate) - Viruses are 1/100000 size of E. coli Infectious cycle overview - Host cell has receptors that recognize specific proteins on virus. Binding=virus enters host cell through endocytosis - Use cell’s ribosomal machinery to make viral proteins that form virions. - Virion: infectious viral particle. Newly formed particle that contains nucleic info and viral proteins. Virion=vehicles to transmit to a next host - Virus use cells energy , mitochon gives energy virus needs to transport vesicles, protein translation machinery Basic viral replication kinetics - Bacteria replicate by binary fission (linearly), diff from viruses (exponentially) - Virus enters, uncoat, release genome, replicate genome to form diff proteins and form infectious virion (takes time) - Adsorption period=attachment. Eclipse period= time when infectivity disappears due to the uncoating - Latent period=replication of genome & protein synthesis. Maturation period=assembly of genome and viral proteins - A cell that takes up a virus and allows the virus to replicate is susceptible (functional receptor for virus) and permissive (allows virus to replicate) - Resistant cell=no receptor, may still support replication though - Cell can be susceptible but not permissive (virus enters but doesn’t replicate no infectious virions) Lab hosts utilized to study infectious cycle - Whole animal hosts (chimps, rats) - Fertilized chicken eggscomposed of multiple cell types, make flu vaccine - Cell culture. Continuous cell lines=immoral cell lines, tumor. Primary cell lines=extracted from living organism, short-lived How to asses infectious cycle - CPE, measure infectivity using plaque or transformation assays, measure virus particles (#) by hemaglutination assay, viral enzyme activity, immunostaining, immunoblotting, sequencing, fluorescent proteins - Infectivity and physical measurements Cytopathic effects (CPE) - Defn: different changed that a virus induces inside a cell. o Ex. cell lysis o Ex. syncytia: fusion of adjacent plasma cell membranesmultinucleated array of cells. CLUMPING o Ex. transformation: cells no longer flat but divide uncontrollably to become piles of round cells  20% of cancers caused by viruses via transformation - Cells become round=hallmark of CPE (cells round up when they die) - Not all viruses give CPEs - Cytopathic viruses kill cells after infections b/c viral burst. Viral burst does not apply to viruses that are not cytopathic Infectivity measurements - A. Cytopathic effects - B. Plaque assay: first used for bacteriophage (virus that infects bacteria) o Agar plate, plaques=area where bacteria have been infected with virus. Replication virus kills bacteria resulting in clear area in bacterial lawn (dots). Agar overlay restricts movement of all the virions released o Can Measure # of infectious particles using dilutions. Avg # of plaques x dilution factor =PFU/mL of original virus suspension o Can purify virus by dilutions - Often, a plaque represents the infectivity from a SINGLE viral particle. This SINGLE particle can initiate infection - Staining cells that are alive with crystal violet=another technique - Are all viral particles infectious? Concept of Particle-to-PFU Ratio: # of virus particles / number of infectious particles o PFU=measure of infectivity or # of infectious particles - C. Transformation assay o Certain viruses do not form plaques but form foci (mountains of cells). Ex Rous Sarcoma Virus (RSV) forms foci. o Can count foci and get foci forming units per/mL Physical measurements - Last option: cells fail to display cytopathic effects and can’t measure infectivity using plaque assays or transformation assays - Measure virus particles (not infectivity but #s since no CPEs). Measure of physical components being expressed ex. proteins - A. Hemaglutination assay o Certain viruses contain proteins that bind to red blood cells. o If a sample contains viruses, they will bind RBC and form a distinct LATTICE which coats the side of the tube o Lack of virus results in RBCs forming a “dot”. No virus=no lattice formation - B. Viral enzyme activity o Open up virus, add radioactively labeled ATPs, proof of presence of retrovirus particles (reverse transcriptase) - C. Immunostaining direct (indicator on antibody which binds to viral antigen) or indirect (indicator on antibody that binds to another antibody that binds to viral antigen) - D. Immunoblotting  measure amount of protein being produced - E. Sequencing useful for low viral abundance genes, exponential growth of gene product - F. Fluorescent proteins by insertion of gene that will be translated into a fluorescent protein adjacent to viral genome Background on the importance of the genome - Hershey-Chase Experimentradioactive phosphorus tags genomefound inside cell Viral classification scheme using the Baltimore method - KEY CONCEPT: Viral genomes must make mRNA; this mRNA must be read by host ribosomes. - Classified viruses with respect to their genomes - Viruses do not follow central dogma. Common goal: production of mRNA to be translated by host cell machinery - + strand = final product just before host ribosomal machinery makes protein - +mRNA strand is ready to be translated, +DNA strand equivalent polarity to +RNA strands - 7 types of genomes (DNA or RNA but not both): o RNA: ss+RNA, ss-RNA, dsRNA, ss+RNA with DNA intermediate o DNA: ssDNA, dsDNA, gapped ds DNA How DNA based genomes get their message across - JC Virus: 80% ppl have it but does nothing unless you have AIDSdegenerative brain diseases - Adenovirusrespiratory and GI tract infections How RNA based genomes get their message across - Host doesn’t have enzyme that is RNA-dependent RNA polym - Thus (-) RNA strands require a virally encoded RNA-dependent RNA polymerase to make mRNA readable by host translation - Rotavirus: most important cause of gastroenteritis worldwide and infant mortality in developing world - Poliovirus: disabling paralysis Genome How It Becomes Enzymes Used Viral Family Specific Notes mRNA Virus dsDNA + RNA DNA-dependent RNA Polyomaviridae(sm JC virus Smaller polym from host genome all) genomes use polym from DNA-dependent RNA Adenovirus host, larger polym from viral genome Adenoviridae(large) Smallpox genomes Poxviridae(large) have viral polym. gapped dsDNA then Viral-associated DNA Hepadnaviruses Hep B (liver Gapped dsDNA ss+RNA polym FILLS in gaps damage) dsDNA also DNA-dependent RNA contains polymerase from host protein and piece of RNA ssDNA dsDNA then Host DNA polymerase Parvoviridae Parvoviruses Single ss+RNA makes dsDNA (cat/dog strands can’t Host RNA polymerase infections) be copied to makes mRNA mRNA dsRNA Make Viral RNA-dependent Reoviridae Rotovirus dsRNA can’t +RNAmRNA RNA pol (in package) be translated uses (-) RNA strand as template New +RNA strand becomes mRNA ss+RNA Ready to go No need for virally Picornaviridae Poliovirus encoded RNA- dependent polym ss-RNA (-)RNA to +RNA Viral polym copes Paramyxoviridae (-)RNA to +RNA Orthomyxoviridae Influenza Virus ss+RNA Make ssDNA Reverse Retroviridae HIV, AIDS ssDNA can’t with DNA from RNA transcriptase=viral RNA- be transcribed intermediat ssDNAdsDNA dependent DNA pol to e make ssDNA Cellular DNA pol makes dsDNA DNA transcription to mRNA - Always have the host DNA dependent polym making the dsDNA Definitions - Subunit=single polypep - Protamer=unit forming capsids or nucleocapsids (subunits together) - Capsid=viral container or shell (protamers together forming a shell) - Virion=complete infectious particle (everything packaged together) - Nucleocapsid=situation where the capsid also contains the genome, capsid attached to genome - Envelope=lipid bilayer membranes enclosing nucleocapsids. Is host derived. (surrounds capsid that is surrounding genome) - The virion must be both stable and unstable. The stability varies during different times of infectious cycle o Fuses with host membrane then uncoats o Stable to allow for host host recognition b/n virion and host cell receptor o Not stable when it enters, uncoating - Capsid pro
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